IMMUNOMODULATORY AGENT AND USES THEREFOR
Disclosed are immunomodulatory agents that are useful for treating or preventing joint damage. More particularly, immunomodulators are disclosed for use in eliciting an antigen-specific tolerogenic response to an aggrecan polypeptide including citrullinated forms thereof to treat or prevent joint damage, including joint damage in subjects with early RA or incipient RA.
This application claims priority to Australian Provisional Application No. 2012901189 entitled “Immunomodulatory Agents and Uses Therefor” filed 23 Mar. 2012, the entire contents of which are incorporated herein by reference.
FIELD OF THE INVENTIONThis invention relates generally to immunomodulatory agents that are useful for treating or preventing joint damage. More particularly, the present invention relates to the use of immune modulators that elicit an antigen-specific tolerogenic response to an aggrecan polypeptide including citrullinated forms thereof to treat or prevent joint damage, including joint damage in subjects with early RA or incipient RA.
BACKGROUND OF THE INVENTIONInflammatory arthritis is a prominent clinical manifestation in diverse autoimmune disorders including rheumatoid arthritis (RA), psoriatic arthritis (PsA), systemic lupus erythematosus (SLE), Sjogren's syndrome, and polymyositis. Most of these patients develop joint deformities on physical examination but typically only RA and PsA patients manifest bone erosions on imaging studies.
The pathogenesis of chronic inflammatory bone diseases, such as RA, is not fully elucidated. Such diseases are accompanied by bone loss around affected joints due to increased osteoclastic resorption, which is mediated largely by increased local production of pro-inflammatory cytokines (Teitelbaum, 2000. Science 289:1504-1508; Goldring and Gravallese, 2000. Arthritis Res. 2(1):33-37). These cytokines can act directly on cells in the osteoclast lineage or indirectly by affecting the production of the essential osteoclast differentiation factor, receptor activator of NF-κB ligand (RANKL), and/or its soluble decoy receptor, osteoprotegrin (OPG), by osteoblast/stromal cells (Hossbauer et al., 2000. J. Bone Min. Res. 15(1):2-12).
RA is a systemic inflammatory disease that affects approximately 0.5 to 1% of the adult population in northern Europe and North America, and a slightly lower proportion in other parts of the world (Alamanos and Drosos, 2005. Autoimmun. Rev. 4: 130-136). It is characterized by chronic inflammation of joint synovial tissue, which ultimately leads to loss of daily function due to chronic pain and fatigue. It is s a chronic inflammatory disease. The majority of patients also experience progressive deterioration of cartilage and bone in the affected joints, which may eventually lead to permanent disability. The long-term prognosis of RA is poor, with approximately 50% of patients experiencing significant functional disability within 10 years from the time of diagnosis.
Several autoantigens are described in RA, including a variety of proteins that become citrullinated in diseased joints. Citrullination is a physiological process of arginine deimination that occurs during apoptosis and inflammation. This process results in modification of arginine-containing proteins, which can give rise to sets of neo-self antigens in individuals bearing at-risk HLA alleles (Vossenaar E R, et al., 2004. Arthritis Res. Ther. 6:107-11). Specific HLA-DR gene variants mapping to amino acids 70-74 of the third hypervariable region of DRβ-chains, are highly associated with RA (Gregersen P K, et al., 1987. Arthritis Rheum. 30:1205-1213). This region encodes a conserved amino acid sequence that forms the fourth anchoring pocket (P4) in the HLA-DR antigen-binding groove. This “shared susceptibility epitope” (SE) is found in multiple RA-associated DR alleles, including DRB1*0401, DRB1*0404 and DRB*0101 in Caucasians (Gregersen P K, et al., 1987, supra). The SE-encoding HLA alleles are particularly associated with ACPA-positive RA (Klareskog L, et al., 2006. Arthritis Rheum. 54:38-46; van Gaalen F A, et al., 2004. Arthritis Rheum. 50:2113-21; Hida S, et al., 2004. J. Autoimmun. 23:141-50; Hill J A, et al., 2003. J. Immunol. 171:538-41).
The SE is highly positively charged, is situated in a region of the DRβ-chain that influences the specificity of the P4 amino acid of the bound ligand and would therefore preferentially bind peptides containing a negatively charged or non-polar amino acid at this position. Citrullination replaces charged arginine amino side-chain groups with an uncharged carbonyl group, and increases the binding affinity of a human vimentin peptide epitope to SE+ HLA DR molecules (Hill J A, et al., 2003, supra; Snir O, et al., 2011. Arthritis Rheum. n/a-n/a).
Approximately 70% of RA patient sera contain anti-citrullinated protein autoantibodies (ACPA) (Meyer O, et al., 2006. Arthritis Res. Ther. 8:R40). This reactivity reflects autoantibody production towards a group of citrullinated autoantigens modified post-translationally, including fibrinogen, vimentin, collagen type II and enolase (Wegner N, et al., 2010. Immunol. Rev. 233:34-54). ACPA develop up to 15 years prior to the onset of RA, with increasing titers and peptide reactivities as disease onset becomes imminent (van de Stadt L A, et al., 2011. Arthritis Rheum. n/a-n/a). Citrullinated proteins have been demonstrated in inflamed tissues in RA, and ACPA are induced in a number of mouse models of inflammatory arthritis (Masson-Bessiere C, et al., 2001. J. Immunol. 166:4177-84; Vossenaar E R, et al., 2003. Arthritis Rheum. 48:2489-500; Kuhn K A, et al., 2006. J. Clin. Invest. 116:961-73; Glant T T, et al., 2011. Arthritis Rheum. 63:1312-1321.). Although citrullination is ubiquitous in response to stress and inflammation, ACPA are highly specific for RA and are associated with more severe joint damage and radiographic outcome (Klareskog L, et al., 2006, supra; van Gaalen F A, et al., 2004, supra; Meyer O, et al., 2006, supra).
Immunization of HLA-DRB1*0401 transgenic mice with citrullinated fibrinogen, but not native fibrinogen, induced inflammatory arthritis characterized by simultaneous B cell and T cell autoreactivity to citrullinated and native HLA-DR-restricted fibrinogen epitopes, which was not present in naïve HLA-DRB1*0401 transgenic mice. The capacity of citrullinated rather than native fibrinogen to induce arthritis suggested that one or more citrullinated neo-self epitopes broke T cell tolerance to corresponding native epitopes, either during priming or resulting from epitope spreading (Hill J A, et al., 2008. J. Exp. Med. 205:967-979). Furthermore, recent studies suggest that delivery of abatacept may restore tolerance towards citrullinated antigens (Yue D, et al., 2010. Arthritis Rheum. 62:2941-2952). Notwithstanding these studies in transgenic mice, citrulline-specific autoreactive T cells have been difficult to demonstrate in RA patients due to the weak proliferative responses made by autoreactive effector memory T cells in vitro. However, several recent papers have shown convincing cytokine responses made by RA patient T cells in response to citrullinated vimentin and aggrecan epitopes (Snir O, et al., 2011, supra; von Delwig A, et al., 2010. Arthritis Rheum. 62:143-149). In the case of vimentin, the immunogenicity of the epitope was dependent on the location of the citrulline modification within the peptide sequence (Snir O, et al., 2011, supra).
The present inventors have now profiled the responses of SE+ healthy controls and RA patients towards a set of citrullinated and un-modified (native) self-antigens, and characterized the responding T cells. Their aim was: (1) to identify citrullinated epitopes that may be of particular relevance in ACPA+ RA; (2) to determine the extent of individual variability among citrullinated autoantigenic T cell responses; and (3) to identify T cells that may contribute to the development of ACPA+ RA.
SUMMARY OF THE INVENTIONThe present invention stems in part from the unexpected discovery that RA patients with long-standing disease are more likely to make a T lymphocyte response to multiple citrullinated autoantigens, whereas patients with recent-onset RA (including those previously untreated) are more likely to respond either to no antigen or only to citrullinated aggrecan. Based on these observations, it is proposed that antigens corresponding in whole, or in part, to aggrecan, including citrullinated aggrecan, will be useful in immunotherapeutic strategies for treating or preventing joint damage, including in subjects with early RA (e.g., when clinical symptoms such as swelling of the joints or pain are not yet present), or in subjects at risk of developing RA (e.g., incipient RA).
Accordingly, in one aspect, the present invention provides methods for treating or preventing joint damage in a subject. These methods generally comprise, consist or consist essentially of eliciting an antigen-specific tolerogenic response to an aggrecan polypeptide (e.g., a citrullinated aggrecan polypeptide, which is also referred to interchangeably herein as “cit-aggrecan” or “cit-agg” polypeptide) in the subject, thereby treating or preventing the joint damage. In some embodiments, the subject has early RA or incipient RA and in illustrative examples of this type, the methods further comprise identifying that the subject has early RA or incipient RA, suitably prior to eliciting the antigen-specific tolerogenic response. In some embodiments, the subject is positive for the shared epitope (SE) and in illustrative examples of this type, the methods further comprise identifying that the subject is positive for SE prior to eliciting the antigen-specific tolerogenic response. Suitably, the subject has or is at risk of developing an immune response including an effector immune response (e.g., an effector T lymphocyte response such as but not limited to a CD4+ effector T lymphocyte) to the aggrecan polypeptide (e.g., a cit-agg polypeptide) or fragments thereof. In non-limiting examples the immune response comprises production (e.g., secretion) of at least one cytokine selected from the group consisting of interleukin-6 (IL-6), interferon-γ (IFN-γ), tumor necrosis factor (TNF), and interleukin-10 (IL-10). In some embodiments, the immune response includes a pro-inflammatory T lymphocyte response, which comprises, consists or consists essentially of production (e.g., secretion) of at least one cytokine selected from the group consisting of IL-6, IFN-γ and TNF. In illustrative examples of this type, the pro-inflammatory T lymphocyte response comprises, consists or consists essentially of production (e.g., secretion) of IL-6. Suitably, the immune response is produced at least in part by CD4+ CD28− T lymphocytes. In some embodiments, the immune response is produced at least in part by CD4+ CD28+ T lymphocytes. In specific embodiments, the immune response is produced at least in part by CD4+ CD28− T lymphocytes and CD4+ CD28+ T lymphocytes.
The antigen-specific tolerogenic response may be achieved using any suitable strategy, illustrative examples of which include:
(1) increasing the number of tolerogenic antigen-presenting cells in the subject, which present a peptide (e.g., an autoantigen) corresponding to a portion of the aggrecan polypeptide (also referred to herein as “aggrecan-specific tolerogenic antigen-presenting cells” or “agg-tolAPC”), wherein the portion is associated with a pro-inflammatory or autoreactive T lymphocyte response to the aggrecan polypeptide (e.g., a cit-agg polypeptide);
(2) inducing anergy or apoptosis in pro-inflammatory or autoreactive T lymphocytes (e.g., effector T lymphocytes) in the subject, which are reactive against the aggrecan polypeptide (e.g., a cit-agg polypeptide) or portion thereof; and
(3) increasing the number of regulatory or suppressor T lymphocytes in the subject, which suppress or otherwise reduce a pro-inflammatory or autoreactive T lymphocyte response to the aggrecan polypeptide.
Thus, in some embodiments, the methods of the present invention comprise, consist or consist essentially of increasing the number of agg-tolAPC in the subject to thereby treat or prevent the joint damage. In some embodiments, the agg-tolAPC stimulate the production of regulatory or suppressor T lymphocytes that suppress or otherwise reduce the pro-inflammatory or autoreactive T lymphocyte response to the aggrecan polypeptide (e.g., a cit-aggrecan polypeptide). Suitably, the regulatory or suppressor T lymphocyte expresses at least one marker (e.g., 1, 2, 3, 4, 5 etc.) of a constitutive regulatory or suppressor T lymphocyte (e.g., at least one marker selected from CD4, CD25, CD62L, GITR, CTLA4) and the transcription factor Forkhead box P3 (FoxP3). Illustrative regulatory or suppressor T lymphocytes include, but are not limited to, CD4+CD25+ regulatory T lymphocytes, Tr1 lymphocytes, Th3 lymphocytes, and CD8+ regulatory T lymphocytes. In specific embodiments, the regulatory T lymphocyte is CD4+CD25+. Non-limiting antigen-presenting cells include dendritic cells, macrophages, Langerhans cells, B lymphocytes and artificial antigen-presenting cells.
The aggrecan-specific tolerogenic antigen-presenting cells may be produced using any suitable strategy. In some embodiments, they are produced by contacting antigen-presenting cells (e.g., dendritic cells, macrophages, Langerhans cells, B cells etc.) with: (1) at least one NF-κB inhibitor in an amount sufficient to inhibit the NF-κB pathway of the antigen-presenting cells; and/or (2) at least one mTOR inhibitor in an amount sufficient to inhibit mTOR of the antigen-presenting cells; and/or (3) at least one Syk inhibitor in an amount sufficient to inhibit the Syk pathway of the antigen-presenting cells, and with an antigenic molecule selected from an antigen that corresponds in whole, or in part, to an aggrecan polypeptide (e.g., a cit-aggrecan polypeptide) or a nucleic acid molecule from which the antigen is expressible, in an amount sufficient for the antigen-presenting cells to present the antigen or a processed form thereof on their surface. In illustrative examples of this type, the methods comprise, consist or consist essentially of co-administering to the subject (e.g., a subject having or at risk of developing joint damage such as a subject with early RA or incipient RA) an NF-κB inhibitor and an antigenic molecule selected from an antigen that corresponds in whole, or in part, to an aggrecan polypeptide (e.g., a cit-aggrecan polypeptide) or a nucleic acid molecule from which the antigen is expressible, wherein the inhibitor is administered in an amount sufficient to inhibit NF-κB activity in an antigen-presenting cell of the subject and wherein the antigenic molecule is administered in an amount sufficient for the NF-κB activity-inhibited antigen-presenting cell to present the antigen or a processed form thereof to the immune system of the subject. In other illustrative examples, the methods comprise, consist or consist essentially of co-administering to the subject (e.g., a subject having or at risk of developing joint damage such as a subject with early RA or incipient RA) an mTOR inhibitor and an antigenic molecule selected from an antigen that corresponds in whole, or in part, to an aggrecan polypeptide (e.g., a cit-aggrecan polypeptide) or a nucleic acid molecule from which the antigen is expressible, wherein the inhibitor is administered in an amount sufficient to inhibit mTOR activity in an antigen-presenting cell of the subject and wherein the antigenic molecule is administered in an amount sufficient for the mTOR activity-inhibited antigen-presenting cell to present the antigen or a processed form thereof to the immune system of the subject. In still other illustrative examples, the methods comprise, consist or consist essentially of co-administering to the subject (e.g., a subject having or at risk of developing joint damage such as a subject with early RA or incipient RA) a Syk inhibitor and an antigenic molecule selected from an antigen that corresponds in whole, or in part, to an aggrecan polypeptide (e.g., a cit-aggrecan polypeptide) or a nucleic acid molecule from which the antigen is expressible, wherein the inhibitor is administered in an amount sufficient to inhibit Syk activity in an antigen-presenting cell of the subject and wherein the antigenic molecule is administered in an amount sufficient for the NF-κB activity-inhibited antigen-presenting cell to present the antigen or a processed form thereof to the immune system of the subject. Suitably, when the antigenic molecule is a nucleic acid molecule from which the antigen is expressible, the nucleic acid molecule is generally in the form of a nucleic acid construct comprising a nucleotide sequence that encodes the antigen and that is operably connected to a regulatory element that is operable in the antigen-presenting cell. In these embodiments, one or more inhibitors (e.g., at least one NF-κB inhibitor and/or at least one mTOR inhibitor and/or at least one Syk inhibitor) and/or the antigenic molecule are generally in a form that is suitable for introduction (e.g., by transformation, internalization, endocytosis or phagocytosis) into the antigen-presenting cells or their precursors, which includes soluble and particulate forms of the inhibitor and/or antigenic molecule.
In some embodiments, the antigen-presenting cells are contacted with at least one inhibitor of the NF-κB pathway, illustrative examples of which are listed in Tables 2, 3A, 3B or 4 infra. In specific embodiments, the NF-κB inhibitor is curcumin or a curcumin derivative.
The aggrecan antigen may comprise, consist or consist essentially of an amino acid sequence corresponding to a putatively full-length citrullinated aggrecan polypeptide including unprocessed, partially processed and mature forms thereof. Alternatively, the aggrecan antigen may comprise, consist or consist essentially of a domain of a citrullinated aggrecan polypeptide such as but not limited to the G1, G2 and G3 domains. In some embodiments, the antigen comprises, consists or consists essentially of an amino acid sequence corresponding to a T cell epitope. In other embodiments, the antigen comprises, consists or consists essentially of a plurality of peptides wherein individual peptides comprise different portions of an amino acid sequence corresponding to an aggrecan polypeptide (e.g., a cit-aggrecan polypeptide). In illustrative examples of this type, the peptides are overlapping peptides.
In related embodiments, the inhibitor (e.g., NF-κB inhibitor, mTOR inhibitor and/or Syk inhibitor) and the antigenic molecule are co-administered in soluble or in particulate form (e.g., the antigenic molecule and the inhibitor are both in soluble form, or one of the antigenic molecule or inhibitor is in soluble form and the other is in particulate form, or the antigenic molecule and the inhibitor are both in particulate form). In specific embodiments, both the inhibitor and the antigenic molecule are co-administered in particulate form. For example, the inhibitor and the antigenic molecule may be contained in one or more particles (e.g., nanoparticles or microparticles such as liposomes and polymeric particles), which are suitably capable of being taken up by an antigen-presenting cell. In specific embodiments, the inhibitor and the antigenic molecule are contained in the same particle. The inhibitor and the antigenic molecule may be administered by injection, by topical application or by the nasal or oral route including sustained-release modes of administration, over a period of time and in amounts which are suitably effective to suppress or otherwise reduce a T lymphocyte response to the aggrecan polypeptide or to ameliorate the symptoms of RA. In specific embodiments, the inhibitor and the antigenic molecule are co-administered subcutaneously.
In other embodiments, the agg-tolAPC are produced by expressing in B lymphocytes a nucleic acid molecule that encodes an antigen that corresponds in whole, or in part, to an aggrecan polypeptide (e.g., a cit-aggrecan polypeptide), wherein the expression of the nucleic acid molecule leads to presentation of the antigen or processed form thereof on the surface of the B lymphocytes. Desirably, in these examples, the nucleic acid molecule further encodes an immunoglobulin (e.g., IgG) or a fragment (e.g., Fv, Fab, Fab′ and F(ab′)2 immunoglobulin fragments, immunoglobulin heavy chain etc.) of an immunoglobulin fused directly or indirectly to the antigen (e.g., adjacent to the C-terminus of the antigen).
In some embodiments, the methods comprise, consist or consist essentially of administering agg-tolAPC or precursors thereof to the subject in an amount effective to suppress or otherwise reduce the pro-inflammatory or autoreactive T lymphocyte response to the aggrecan polypeptide (e.g., a cit-aggrecan polypeptide) or to inhibit the development of that response. In illustrative examples of this type, the agg-tolAPC or their precursors are produced by harvesting antigen-presenting cells or antigen-presenting cell precursors from the subject or from a histocompatible donor and exposing them ex vivo to: (A1) at least one NF-κB inhibitor in an amount sufficient to inhibit the NF-κB pathway of the antigen-presenting cells; and/or (A2) at least one mTOR inhibitor in amounts sufficient to inhibit mTOR of the antigen-presenting cells; and/or (A3) at least one Syk inhibitor in an amount sufficient to inhibit the Syk pathway of the antigen-presenting cells, and to (B) an antigenic molecule selected from an antigen that corresponds in whole, or in part, to an aggrecan polypeptide (e.g., a cit-aggrecan polypeptide) or a nucleic acid molecule from which the antigen is expressible, in an amount sufficient for the antigen-presenting cells or their precursors to present the antigen or a processed form thereof to the immune system of the subject.
In other illustrative examples, the agg-tolAPC or their precursors are produced by harvesting B lymphocytes or B lymphocyte precursors from the subject or from a histocompatible donor and introducing into them ex vivo a nucleic acid molecule which is operably connected to a regulatory element that is operable in the B lymphocytes and which encodes an antigen that corresponds in whole, or in part, to an aggrecan polypeptide (e.g., a cit-aggrecan polypeptide), wherein expression of the nucleic acid molecule leads to presentation of the antigen or processed form thereof on the surface of the B lymphocytes. In specific embodiments, the nucleic acid molecule further encodes an immunoglobulin or an immunoglobulin fragment fused directly or indirectly to the antigen. Suitably, when precursors are used, the precursors are cultured for a time and under conditions sufficient to differentiate antigen-presenting cells from the precursors.
In other embodiments, the methods comprise, consist or consist essentially of administering to the subject a MHC-peptide complex consisting essentially of an antigen that corresponds in whole, or in part, to an aggrecan polypeptide (e.g., a cit-aggrecan polypeptide) and an isolated (e.g., soluble) MHC component having an antigen-binding site, wherein the antigen is associated with the antigen-binding site.
In still other embodiments, the methods comprise, consist or consist essentially of administering to the subject a chimeric construct comprising an immunomodulatory peptide and an immune- or T cell-binding ligand (I/TCBL), wherein the immunomodulatory peptide comprises, consists or consists essentially of an amino acid sequence corresponding to a portion (e.g., an autoantigen) of an aggrecan polypeptide (e.g., a cit-aggrecan polypeptide), which is suitably associated with rheumatoid arthritis (RA), and which binds to an antigen receptor (e.g., T cell receptor) on pro-inflammatory or autoreactive T lymphocytes, and wherein the I/TCBL binds to a class or subclass of T cell selected from the group consisting of helper T cells, suppressor T cells and cytotoxic T cells and modulates T cell activity. Suitably, the I/TCBL comprises at least a portion of a molecule selected from a MHC class I molecule, a MHC class II molecule, an accessory molecule such as β2-microglobulin, lymphocyte function associated molecule-3 (LFA-3), the Fc region of the heavy chain of an immunoglobulin molecule, Ia+ molecules, an antibody to CD2, an antibody to CD3, an antibody to CD4, an antibody to CD8, an antibody to lectin, a lymphokine.
In other embodiments, the methods comprise, consist or consist essentially of administering to the subject an altered peptide ligand (APL) that comprises, consists or consists essentially of an amino acid sequence corresponding to a portion (e.g., an autoantigen) of an aggrecan polypeptide (e.g., a cit-aggrecan polypeptide), which is suitably associated with rheumatoid arthritis (RA), and which binds to an antigen receptor (e.g., T cell receptor) on pro-inflammatory or autoreactive T lymphocytes, wherein the APL amino acid sequence is distinguished from the amino acid sequence of the portion by at least one amino acid substitution, deletion or addition, interferes with normal signaling through the antigen receptor and has at least one activity selected from: (i) antagonizing the response of the T lymphocytes to the aggrecan polypeptide (e.g., a cit-aggrecan polypeptide), (ii) inducing anergy in aggrecan- or citrullinated aggrecan-specific T lymphocytes, (iii) inducing apoptosis in aggrecan- or citrullinated aggrecan-specific T lymphocytes, (iv) stimulating or inducing a aggrecan- or citrullinated aggrecan-specific Th2 immune response, (v) suppressing development of a aggrecan- or citrullinated aggrecan-specific Th1 immune response including suppressing the production of pro-inflammatory cytokines, (vi) stimulating activation of aggrecan- or citrullinated aggrecan-specific regulatory lymphocytes (e.g., T regulatory lymphocytes (Treg)), or (vii) preventing or inhibiting the activation of aggrecan- or citrullinated aggrecan-specific antigen-presenting cells by an inflammatory stimulus.
In related aspects, the invention extends to the use of an agent for treating or preventing joint disease (e.g., early RA or incipient RA) or in the manufacture of a medicament for treating or preventing joint disease (e.g., early RA or incipient RA), wherein the agent is selected from: (1) an inhibitor of the NF-κB pathway and an antigenic molecule selected from an antigen that corresponds in whole, or in part, to an aggrecan polypeptide (e.g., a cit-aggrecan polypeptide) or a nucleic acid molecule from which the antigen is expressible, as broadly described above; (2) an mTOR inhibitor and an antigenic molecule selected from an antigen that corresponds in whole, or in part, to an aggrecan polypeptide (e.g., a cit-aggrecan polypeptide) or a nucleic acid molecule from which the antigen is expressible, as broadly described above; (3) an inhibitor of the Syk pathway and an antigenic molecule selected from an antigen that corresponds in whole, or in part, to an aggrecan polypeptide (e.g., a cit-aggrecan polypeptide) or a nucleic acid molecule from which the antigen is expressible, as broadly described above; (4) a nucleic acid molecule that encodes an antigen that corresponds in whole, or in part, to an aggrecan polypeptide (e.g., a cit-aggrecan polypeptide) for introduction into B lymphocytes, as broadly described above; (5) a MHC-peptide complex consisting essentially of an antigen that corresponds in whole, or in part, to an aggrecan polypeptide (e.g., a cit-aggrecan polypeptide) and an isolated (e.g., soluble) MHC component having an antigen-binding site, wherein the antigen is associated with the antigen-binding site, as broadly described above; (6) a chimeric construct as broadly described above; (7) aggrecan-specific tolerogenic antigen-presenting cells as broadly described above; or (8) an APL that comprises, consists or consists essentially of an amino acid sequence corresponding to a portion of an aggrecan polypeptide (e.g., a cit-aggrecan polypeptide), as broadly described above.
Still another aspect of the present invention relates to compositions, which are suitably useful for treating or preventing joint damage in a subject. These compositions generally comprise, consist or consist essentially of an agent selected from: (1) an inhibitor of the NF-κB pathway and an antigenic molecule selected from an antigen that corresponds in whole, or in part, to an aggrecan polypeptide (e.g., a cit-aggrecan polypeptide) or a nucleic acid molecule from which the antigen is expressible, as broadly described above; (2) an mTOR inhibitor and an antigenic molecule selected from an antigen that corresponds in whole, or in part, to an aggrecan polypeptide (e.g., a cit-aggrecan polypeptide) or a nucleic acid molecule from which the antigen is expressible, as broadly described above; (3) an inhibitor of the Syk pathway and an antigenic molecule selected from an antigen that corresponds in whole, or in part, to an aggrecan polypeptide (e.g., a cit-aggrecan polypeptide) or a nucleic acid molecule from which the antigen is expressible, as broadly described above; (4) a nucleic acid molecule that encodes an antigen that corresponds in whole, or in part, to an aggrecan polypeptide (e.g., a cit-aggrecan polypeptide) for introduction into B lymphocytes, as broadly described above; (5) a MHC-peptide complex consisting essentially of an antigen that corresponds in whole, or in part, to an aggrecan polypeptide (e.g., a cit-aggrecan polypeptide) and an isolated (e.g., soluble) MHC component having an antigen-binding site, wherein the antigen is associated with the antigen-binding site, as broadly described above; (6) a chimeric construct as broadly described above; (7) aggrecan-specific tolerogenic antigen-presenting cells as broadly described above; or (8) an APL that comprises, consists or consists essentially of an amino acid sequence corresponding to a portion of an aggrecan polypeptide (e.g., a cit-aggrecan polypeptide), as broadly described above.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.
The articles “a” and “an” are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.
The term “about” is used herein to refer to conditions (e.g., amounts, concentrations, time etc.) that vary by as much as 15%, and suitably by as much as 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% to a specified condition.
The terms “administration concurrently” or “administering concurrently” or “co-administering” and the like refer to the administration of a single composition containing two or more actives, or the administration of each active as separate compositions and/or delivered by separate routes either contemporaneously or simultaneously or sequentially within a short enough period of time that the effective result is equivalent to that obtained when all such actives are administered as a single composition. By “simultaneously” is meant that the active agents are administered at substantially the same time, and desirably together in the same formulation. By “contemporaneously” it is meant that the active agents are administered closely in time, e.g., one agent is administered within from about one minute to within about one day before or after another. Any contemporaneous time is useful. However, it will often be the case that when not administered simultaneously, the agents will be administered within about one minute to within about eight hours and preferably within less than about one to about four hours. When administered contemporaneously, the agents are suitably administered at the same site on the subject. The term “same site” includes the exact location, but can be within about 0.5 to about 15 centimeters, preferably from within about 0.5 to about 5 centimeters. The term “separately” as used herein means that the agents are administered at an interval, for example at an interval of about a day to several weeks or months. The active agents may be administered in either order. The term “sequentially” as used herein means that the agents are administered in sequence, for example at an interval or intervals of minutes, hours, days or weeks. If appropriate the active agents may be administered in a regular repeating cycle.
The term “anergy” as used herein refers to a suppressed response, or a state of non-responsiveness, to a specified antigen or group of antigens by an immune system. For example, T lymphocytes and B lymphocytes are anergic when they cannot respond to their specific antigen under optimal conditions of stimulation.
By “antigen” is meant all, or part of, a protein, peptide, or other molecule or macromolecule capable of eliciting an immune response in a vertebrate animal, especially a mammal. Such antigens are also reactive with antibodies from animals immunized with that protein, peptide, or other molecule or macromolecule.
By “antigen-binding molecule” is meant a molecule that has binding affinity for a target antigen. It will be understood that this term extends to immunoglobulins, immunoglobulin fragments and non-immunoglobulin derived protein frameworks that exhibit antigen-binding activity.
By “autologous” is meant something (e.g., cells, tissues etc.) derived from the same organism.
The term “allogeneic” as used herein refers to cells, tissues, organisms etc. that are of different genetic constitution.
By “alloantigen” is meant an antigen found only in some members of a species, such as blood group antigens. By contrast a “xenoantigen” refers to an antigen that is present in members of one species but not members of another. Correspondingly, an “allograft” is a graft between members of the same species and a “xenograft” is a graft between members of a different species.
The term “biological sample” as used herein refers to a sample that may be extracted, untreated, treated, diluted or concentrated from a subject. The biological sample may include a biological fluid such as whole blood, serum, plasma, saliva, urine, sweat, ascitic fluid, peritoneal fluid, synovial fluid, amniotic fluid, cerebrospinal fluid, tissue biopsy, and the like. In certain embodiments, the biological sample is selected from synovial fluid and blood, including peripheral blood.
“Clinical improvement” refers to prevention of further progress of RA or joint damage or any improvement in RA or joint damage as a result of treatment, as determined by various testing, including radiographic testing. Thus, clinical improvement may, for example, be determined by assessing the number of tender or swollen joints, performing the Disease Activity Score (DAS) or American College of Rheumatology (ACR) score, performing a global clinical assessment of the subject, assessing erythrocyte sedimentation rate, or assessing the amount of C-reactive protein level.
Throughout this specification, unless the context requires otherwise, the words “comprise,” “comprises” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. Thus, use of the term “comprising” and the like indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present. By “consisting of” is meant including, and limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of” is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of” indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.
By “corresponds to” or “corresponding to” is meant an antigen that encodes an amino acid sequence that displays substantial similarity to an amino acid sequence in a target antigen. In general the antigen will display at least about 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% similarity to at least a portion of the target antigen.
“Citrulline” and “Cit” refers to 2-amino-5-(carbamoylamino)pentanoic acid and is an α-amino acid with formula: H2NC(O)NH(CH2)3CH(NH2)CO2H.
The expression “effective amount” refers to an amount of an agent or medicament, either in a single dose or as part of a series, which is effective for treating or preventing RA or joint damage. This would include an amount that is effective in achieving a reduction in RA or joint damage as compared to baseline prior to administration of such amount as determined, e.g., by radiographic or other testing. The effective amount will vary depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated, the formulation of the composition, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
As used herein, “gene therapy” refers to the insertion, ex vivo or in vivo, of a gene or genes into individual cells or groups of cells (such as tissues or organs), whereby expression of the gene or genes in the cells or groups of cells provides a therapeutic effect. Such “therapeutic genes” are generally delivered using a vector. Cells targeted by gene therapy can be either somatic cells or germ cells or cell lines. Gene therapy includes and encompasses the use of vectors to deliver, either ex vivo or in vivo, a gene that requires overexpression or ectopic expression in a cell or group of cells. The vector can facilitate integration of the new gene in the nucleus or can lead to episomal expression of that gene.
“Immune effector cells” refers to cells capable of binding an antigen and which mediate an immune response selective for the antigen. These cells include, but are not limited to, T cells (T lymphocytes), B cells (B lymphocytes), monocytes, macrophages, natural killer (NK) cells and cytotoxic T lymphocytes (CTLs), for example CTL lines, CTL clones, and CTLs from tumor, inflammatory, or other infiltrates
Reference herein to “immuno-interactive” includes reference to any interaction, reaction, or other form of association between molecules and in particular where one of the molecules is, or mimics, a component of the immune system.
“Joint damage” is used herein in the broadest sense and refers to damage or partial or complete destruction to any part of one or more joints, including the connective tissue and cartilage, where damage includes structural and/or functional damage of any cause, and may or may not cause joint pain/arthralgia. It includes, without limitation, joint damage associated with or resulting from inflammatory joint disease as well as non-inflammatory joint disease. This damage may be caused by any condition, such as an autoimmune disease, especially arthritis, and most especially RA. Exemplary conditions of this type include acute and chronic arthritis, RA including juvenile-onset RA, juvenile idiopathic arthritis (JIA), or juvenile RA (JRA), and stages such as rheumatoid synovitis, gout or gouty arthritis, acute immunological arthritis, chronic inflammatory arthritis, degenerative arthritis, type II collagen-induced arthritis, infectious arthritis, septic arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, vertebral arthritis, osteoarthritis, arthritis chronica progrediente, arthritis deformans, polyarthritis chronica primaria, reactive arthritis, menopausal arthritis, estrogen-depletion arthritis, and ankylosing spondylitis/rheumatoid spondylitis), rheumatic autoimmune disease other than RA, and significant systemic involvement secondary to RA (including but not limited to vasculitis, pulmonary fibrosis, or Felty's syndrome). For purposes herein, joints are points of contact between elements of a skeleton (of a vertebrate such as an animal) with the parts that surround and support it and include, but are not limited to, for example, hips, joints between the vertebrae of the spine, joints between the spine and pelvis (sacroiliac joints), joints where the tendons and ligaments attach to bones, joints between the ribs and spine, shoulders, knees, feet, elbows, hands, fingers, ankles and toes, but especially joints in the hands and feet.
Reference herein to a “level or functional activity” in the context of a gene expression product (e.g., a protein or a transcript) produced by a specified cell is to be taken in its broadest sense and includes a level or functional activity of the expression product that is produced in a single cell or in a plurality or population of cells. In the latter case, therefore, it will be understood that the phrase will encompass a mean level or functional activity of the protein produced by a plurality or population of cells.
By “pharmaceutically-acceptable carrier” is meant a solid or liquid filler, diluent or encapsulating substance that may be safely used in topical or systemic administration.
The terms “polyarthritis” and “synovitis” are used interchangeably herein to refer to inflammation, i.e., swelling, tenderness, or warmth, at one or more joints of a subject.
“Polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non-naturally occurring amino acid, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally-occurring amino acid polymers.
“Prevention” or “prophylaxis,” as used herein, refers to prophylactic or preventative measures. Those in need of prevention or prophylaxis include those in whom the RA or joint damage is to be prevented, and in some embodiments, may be predisposed or susceptible to the RA or joint damage e.g. individuals with a family history of RA, or individuals bearing the HLA-SE and anti-citrullinated peptide antibodies but not meeting ACR/EULAR 2010 criteria for RA. Prevention or prophylaxis is successful herein if the development of RA or joint damage is completely or partially prevented or slowed down, as compared to the development of RA or joint or structural damage in the absence of tolerogenesis.
By “regulatory lymphocyte” is meant a lymphocyte that is involved in regulating or suppressing responses and actions of other cells, especially of other immune cells such as B lymphocytes, T helper and innate (including NKT and gamma-delta T) lymphocytes.
As used herein, “rheumatoid arthritis” or “RA refers to a recognized disease state that may be diagnosed according to the 2000 revised American Rheumatoid Association criteria for the classification of RA, or any similar criteria. The term includes not only active and early RA, but also incipient RA, as defined below. Physiological indicators of RA include symmetric joint swelling, which is characteristic though not invariable in RA. Fusiform swelling of the proximal interphalangeal (PIP) joints of the hands as well as metacarpophalangeal (MCP), wrists, elbows, knees, ankles, and metatarsophalangeal (MTP) joints are commonly affected and swelling is easily detected. Pain on passive motion is the most sensitive test for joint inflammation, and inflammation and structural deformity often limits the range of motion for the affected joint. Typical visible changes include ulnar deviation of the fingers at the MCP joints, hyperextension, or hyperflexion of the MCP and PIP joints, flexion contractures of the elbows, and subluxation of the carpal bones and toes. RA includes, for example, juvenile-onset RA, juvenile idiopathic arthritis (JIA), or juvenile RA (JRA). A patient with “active rheumatoid arthritis” means a patient with active and not latent symptoms of RA. Subjects with “early rheumatoid arthritis” are those subjects who are diagnosed as having ‘definite’ RA for no longer than four years, no longer than three years, no longer than two years, no longer than one year, no longer than six months, wherein definite RA is diagnosed when the subject's clinical parameters provide a score of ≧6/10 according to the revised 2010 American College of Rheumatology (ACR)/European League Against Rheumatism (EULAR) criteria for classification of RA (Aletaha et al., 2010. Arthritis & Rheumatism 62(9):2569-2581, which is hereby incorporated by reference herein in its entirety). Patients with “incipient RA” have early polyarthritis (synovitis) that does not fully meet ACR/EULAR criteria for a diagnosis of definite RA (e.g., a score of <6/10). In specific examples, the polyarthritis associates with the presence of RA-specific prognostic biomarkers such as anti-cyclic citrullinated peptide (CCP) antibodies and SE+.
“Shared epitope” or “SE” or “rheumatoid epitope,” as used herein, means the sequence motifs in residues 70 to 74 of the third hypervariable region of the HLA-DRB1 chain encoded by the HLA-DRB1*0401, *0404/0408, *0405, *0409, *0410, *0413, *0416, *0101, *0102, *0104, *1001, *1402, and *1406 alleles in the predisposition to RA. Specifically, the sequence motifs are characterized by the amino acid coding sequence QKRAA (SEQ ID NO: 46) or QRRAA (SEQ ID NO: 47) or RRRAA (SEQ ID NO: 48) in the third hypervariable region, encompassing amino acid residues 70 to 74 of the HLA-DRB1 chain of the major histocompatibility complex class II molecule. Because DNA typing examines the alleles at a given locus, the name of the locus precedes the designation of the specific allele (with the two terms separated by an asterisk); for example, HLA-DRB1*0401 refers to the 0401 allele of the HLA-DRB1 locus. One particular HLA-DR specificity is encoded by several HLA-DRB1 alleles in conjunction with the product of the HLA-DRA1 locus; for example, more than 11 HLA-DRB1 alleles (HLA-DRB1*0401 to *0411) can encode the B chain of the HLA-DR4 specificity. For purposes herein, responsiveness to treatment of RA with an aggrecan-specific tolerogenic therapy is positively correlated with the incidence or presence of this genetic biomarker in patients with alleles for SE that are homozygous or heterozygous.
The terms “subject,” “patient” or “individual,” which are used interchangeably herein, refer to any subject, particularly a vertebrate subject, and even more particularly a mammalian subject, for whom therapy or prophylaxis is desired. Suitable vertebrate animals that fall within the scope of the invention include, but are not restricted to, any member of the subphylum Chordata including humans as well as non-human primates, rodents (e.g., mice rats, guinea pigs), lagomorphs (e.g., rabbits, hares), bovines (e.g., cattle), ovines (e.g., sheep), caprines (e.g., goats), porcines (e.g., pigs), equines (e.g., horses), canines (e.g., dogs), felines (e.g., cats), avians (e.g., chickens, turkeys, ducks, geese, companion birds such as canaries, budgerigars etc.), marine mammals (e.g., dolphins, whales), reptiles (snakes, frogs, lizards etc.), and fish. In specific embodiments, the “subject,” “patient” or “individual” is a human in need of treatment or prophylaxis of joint damage, including in subjects with early RA, or in subjects at risk of developing RA (e.g., incipient RA). In specific embodiments, the terms “subject,” “patient” or “individual” refer to any single human subject, including a patient, eligible for treatment who is experiencing or has experienced one or more signs, symptoms, or other indicators of RA or joint damage, whether, for example, newly diagnosed or previously diagnosed and now experiencing a recurrence or relapse, or is at risk for RA or joint damage, no matter the cause. Intended to be included as a “subject,” “patient” or “individual” are any subjects involved in clinical research trials not showing any clinical sign of disease, or subjects involved in epidemiological studies, or subjects once used as controls. The “subject,” “patient” or “individual” may have been previously treated with a medicament for RA or joint damage, or not so treated.
By “suppression,” “suppressing” and the like is meant any attenuation or regulation of an immune response, including B-lymphocyte and T lymphocyte immune responses, to an antigen or group of antigens. In some embodiments, the attenuation is mediated at least in part by suppressor T lymphocytes (e. g., CD4+CD25+ regulatory T lymphocytes).
As used herein, the term “surfactant” refers to any agent, which preferentially absorbs to an interface between two immiscible phases, such as the interface between water and an organic polymer solution, a water/air interface or organic solvent/air interface. Surfactants generally possess a hydrophilic moiety and a lipophilic moiety; such that, upon absorbing to microparticles, they tend to present moieties to the external environment that do not attract similarly coated particles, thus reducing particle agglomeration. Surfactants may also promote absorption of a therapeutic or diagnostic agent and increase bioavailability of the agent.
As used herein, a particle “incorporating a surfactant” refers to a particle with a surfactant on at least the surface of the particle. The surfactant may be incorporated throughout the particle and on the surface during particle formation, or may be coated on the particle after particle formation. The surfactant can be coated on the particle surface by adsorption, ionic or covalent attachment, or physically “entrapped” by the surrounding matrix. The surfactant can be, for example, incorporated into controlled release particles, such as polymeric microspheres.
A “symptom” of RA or joint damage is any morbid phenomenon or departure from the normal in structure, function, or sensation, experienced by the subject and indicative of RA or joint damage, such as those noted above, including tender or swollen joints.
“Total modified Sharp score” means a score obtained for assessment of radiographs using the method according to Sharp, as modified by Genant (1983, Am. J. Med., 30:35-47). The primary assessment will be the change in the total Sharp-Genant score from screening. The Sharp-Genant score combines an erosion score and a joint space narrowing score of both hands and feet. Joint damage is measured in this test scoring by a mean change of less than the score at baseline (when patient is screened or tested before first administration of the tolerogenic composition as disclosed herein).
As used herein, the term “transduction” or “transduced” to the introduction of foreign or exogenous nucleic acid, usually DNA, into a cell and includes “stable transduction” and “transient transduction.” “Stable transduction” or “stably transduced” refers to the introduction and integration of foreign or exogenous nucleic acid into the genome of the transduced cell. The term “stable transductant” refers to a cell that has stably integrated foreign DNA into the genomic DNA. Conversely, the term “transient transduction” or “transiently transduced” refers to the introduction of foreign or exogenous nucleic acid into a cell where the foreign or exogenous DNA fails to integrate into the genome of the transduced cell. The foreign or exogenous DNA persists in the nucleus of the transducted cell for several days. During this time the foreign or exogenous DNA is subject to the regulatory controls that govern the expression of endogenous genes in the chromosomes. The term “transient transductant” refers to cells that have taken up foreign or exogenous DNA but have failed to integrate this DNA.
“Treatment” of a subject herein refers to therapeutic treatment. Those in need of treatment include those already with RA or joint damage as well as those in whom the progress of RA or joint damage is to be prevented. Hence, the subject may have been diagnosed as having the RA or joint damage or may have RA or joint symptoms or damage that is likely to progress in the absence of treatment. Alternatively the subject may be symptom-free but has risk factors for development of RA e.g., positive family history and/or presence of autoantibodies such as ACPA or rheumatoid factor or evidence of an inflammatory phenotype in peripheral blood. Treatment is successful herein if the RA or joint damage is alleviated or healed, or progression of RA or joint damage, including its signs and symptoms and/or structural damage, is halted or slowed down as compared to the condition of the subject prior to administration. Successful treatment further includes complete or partial prevention of the development of joint or structural damage. For purposes herein, slowing down or reducing RA or joint damage or the progression of joint damage is the same as arrest, decrease, or reversal of the RA or joint damage.
The terms “wild-type” and “normal” are used interchangeably to refer to the phenotype that is characteristic of most of the members of the species occurring naturally and contrast for example with the phenotype of a mutant.
As used herein, underscoring or italicizing the name of a gene shall indicate the gene, in contrast to its protein product, which is indicated by the name of the gene in the absence of any underscoring or italicizing. For example, “ACAN” shall mean the ACAN gene (i.e., the gene encoding aggrecan), whereas “ACAN” shall indicate the protein product or products generated from transcription and translation and alternative splicing of the “ACAN” gene.
2. AbbreviationsThe following abbreviations are used throughout the application:
-
- aa=amino acid(s)
- ACAN=aggrecan gene
- ACAN=aggrecan polypeptide
- ACPA=anti-citrullinated peptide antibodies
- agg-tolAPC=aggrecan-specific antigen-presenting cells
- APC=antigen-presenting cell(s)
- cit-agg=citrullinated aggrecan
- d=Day
- GM-CSF=granulocyte macrophage colony-stimulating factor
- h=Hour
- IL-2=interleukin 2
- IL-3=interleukin 3
- IL-6=interleukin 6
- IL-10=interleukin 10
- IL-12=interleukin 12
- IL-17=interleukin 17
- kb=kilobase(s) or kilobase pair(s)
- kDa=kilodalton(s)
- nt=Nucleotide
- nts=Nucleotides
- PB=peripheral blood
- PBMC=peripheral blood mononuclear cells
- RA=rheumatoid arthritis
- s=seconds
- SE=shared epitope
- TCR=T cell receptor
- tolAPC=tolerogenic antigen-presenting cells
- TNF=tumor necrosis factor
The present invention is based in part on the finding that patients with early RA are more likely to make a T lymphocyte response, including a pro-inflammatory T lymphocyte response, to no autoantigen or to citrullinated aggrecan alone, while patients with longstanding RA are more likely to make a T lymphocyte response, including a pro-inflammatory T lymphocyte response, to multiple autoantigens. These results suggest that T lymphocyte responses to citrullinated aggrecan, including pro-inflammatory T lymphocyte responses, precede heterogeneous maturation of the T lymphocyte response to other citrullinated self-epitopes as RA progresses. It was also found that patients with RA of any disease duration were more likely to respond to citrullinated aggrecan than to other citrullinated self-epitopes. Based on these findings, the present inventors propose that antigens that correspond in whole, or in part, to an aggrecan polypeptide, including citrullinated forms thereof, will allow earlier therapeutic and/or prophylactic intervention of joint damage in affected or predisposed subjects including subjects with early RA and subjects at risk of developing RA, suitably before the disease progresses to the chronic and debilitating form of RA, and that therapeutic application of citrullinated aggrecan antigens will provide broader coverage of the RA population as a whole than of other citrullinated antigens.
The present invention thus provides methods for treating or preventing joint damage in a subject through antigen-specific suppression of an immune response (also referred to herein as an “antigen-specific tolerogenic response”) to an aggrecan polypeptide.
3.1 Aggrecan-Specific Immune Response and Affected SubjectsThe aggrecan polypeptide is typically citrullinated in which at least one arginine residue in the aggrecan sequence is converted to citrulline. If desired, the presence of citrullinated aggrecan polypeptide may be determined using assays that directly detect citrullinated aggrecan, illustrative examples of which include mass spectroscopic analysis, as described for example by Goëb et al. (2009, Arthritis Research & Therapy 11:R38), Stensland et al. (2009, Rapid Commun Mass Spectrum. 23(17):2754-2762), Hermansson et al. (2010, Proteomics Clin Appl. 4(5):511-518), van Beers et al. (2010, Arthritis Res Ther. 12(6):R219), and De Ceuleneer et al. (2011, Rapid Commun Mass Spectrum. 25(11):1536-1542), which are hereby incorporated by reference herein in their entirety. Alternatively, immunoassays may be employed, which use for example anti-modified citrulline antibodies for detecting citrulline residues in an antigen of interest and optionally antigen-specific antibodies for detecting the antigen of interest itself (i.e., whether citrullinated or not). Non-limiting immunoassays of this type are described by Tilleman et al. (2008, Rheumatology 47:597-604), Tabushi et al. (2008, Annals of Clinical Biochemistry 45:413-417) and in US Patent Application 2011/0244492, which are hereby incorporated by reference herein in their entirety. In other examples, the presence of citrullinated aggrecan may be determined indirectly by detecting ACPA specific for citrullinated aggrecan. Representative assays of this type may be set up by using one or more peptides corresponding to a citrullinated aggrecan polypeptide (cit-agg-specific peptides) or a protein corresponding to citrullinated aggrecan polypeptide as antigen, and detecting the binding of cit-agg-specific ACPA comprised in a sample to the peptide antigen by appropriate means. ACPA may be detected by homogeneous assay formats (e.g., by agglutination of latex particles coated with cit-agg peptide), by a heterogeneous immunoassay (e.g., based on directly or indirectly coating cit-agg peptide to a solid phase, incubating the solid phase with a sample known or suspected to comprise cit-agg-specific ACPA under conditions allowing for binding of ACPA antibodies to peptide antigen, and directly or indirectly detecting the ACPA bound) or using a double-antigen bridge assay, in which cit-agg peptide is used both at the solid-phase side as well as at the detection side of this immunoassay. Non-limiting antigen-specific ACPA assays of this type are described for example in Wegner et al. (2010, supra) and bibliographic references listed therein, which are hereby incorporated by reference herein in their entirety.
In specific embodiments, the subject is identified as having an RA stratification selected from early RA or incipient RA, as defined herein, and in illustrative examples of this type, the methods further comprise identifying that the subject has the RA stratification prior to eliciting the antigen-specific tolerogenic response. The RA stratification may be determined using any suitable clinical parameters known to practitioners in the art, non-limiting examples of which include to: 1) age; 2) gender; 3) distribution of involved joints; 4) duration of morning stiffness; 5) number of tender joints (e.g., positive squeeze across metacarpophalangeal or metatarsophalangeal joints); 6) number of swollen joints; 7) joint pain; 8) previous episodes; 9) family history of RA; 10) the presence or absence of systemic flu-like features and fatigue; 11) symmetrical joint involvement in hands and/or feet; 12) erythrocyte sedimentation rate; 13) level of C-reactive protein; 14) level of high sensitivity C-reactive protein; 15) level of rheumatoid factor (RF); 16) presence, absence or level of ACPA antibody; 17) presence, absence or level of antibodies to mutated citrullinated vimentin (anti-MCV antibody); 18) presence or absence of SE; 19) presence, absence or level of the J protein, Hdj2 (e.g., in synovial fluid; 20) rheumatoid nodules; or 21) radiographic changes. In specific embodiments, the presence or absence of RA is assessed using the 2010 ACR/EULAR classification criteria for RA as disclosed by Aletaha et al. (2010, supra), which are summarized in the Table 1.
The symbols *, †, ‡, §, #, **, ††, ‡‡ and §§ have the same meaning as in Aletaha et al. (2010, supra).
In illustrative examples, the subject is identified as having early RA when the subject is diagnosed as having ‘definite’ RA for no longer than four years, no longer than three years, no longer than two years, no longer than one year, no longer than six months, wherein definite RA is identified when the subject's assessed clinical parameters provide a score of ≧6/10 according to the 2010 ACR/EULAR criteria. In other illustrative examples, the subject is identified as having incipient RA when the subject has polyarthritis (synovitis) but does not fully meet ACR/EULAR criteria for a diagnosis of definite RA (e.g., a score of <6/10). In specific examples, the polyarthritis associates with the presence of RA-specific prognostic biomarkers such as ACPA antibodies and shared epitope positive (SE+). Subjects with incipient RA include ACPA antibody-positive patients who present with polyarthritis, and do not yet have a diagnosis of definite RA, but are at high risk for going on to develop definite RA according to the 2010 ACR/EULAR criteria (e.g., 95% probability).
In specific embodiments, the subject is SE+. Shared epitope positivity can be tested using any suitable assay that identifies the sequence motifs in residues 70 to 74 of the third hypervariable region of the HLA-DRB1 chain of the major histocompatibility complex class II molecule, as predisposing the subject to RA. For instance, the sequence motifs may be characterized by the amino acid coding sequence QKRAA (SEQ ID NO: 46) or QRRAA (SEQ ID NO: 47) or RRRAA (SEQ ID NO: 48), which are encoded for example by the HLA-DRB1*0401, *0404/0408, *0405, *0409, *0410, *0413, *0416, *0101, *0102, *0104, *1001, *1402, and *1406 alleles. The assays may involve genotyping using for example sequence analysis, polymerase chain reaction (PCR) typing or oligonucleotide probe hybridization (e.g., reverse hybridization) and immunoassays as known in the art. In these embodiments, the methods may further comprise identifying that the subject is SE+ prior to eliciting the antigen-specific tolerogenic response.
The present inventors have also found that the T lymphocyte (e.g., CD4+ T lymphocyte) response to citrullinated self-epitopes, including citrullinated aggrecan, in subjects with early RA or at risk of developing RA, includes the production (e.g., secretion) of at least 1, 2, 3 or cytokine(s). In specific embodiments, the cytokine(s) is (are) selected from IL-6, IFN-γ, TNF and IL-10. The T lymphocyte response largely includes a pro-inflammatory or autoreactive T lymphocyte response which is characterized by the production (e.g., secretion) of at least 1, 2 or 3 pro-inflammatory (e.g., Th1 or Th17) cytokine(s). In specific embodiments, the pro-inflammatory cytokine(s) is (are) selected from IL-6, IFN-γ and TNF. Thus, in some embodiments, the methods of the present invention may further comprise identifying that the subject produces a pro-inflammatory T lymphocyte response (e.g., the production of at least 1, 2 or 3 pro-inflammatory cytokine selected from IL-6, IFN-γ and TNF) prior to eliciting the antigen-specific tolerogenic response. However, the present inventors have also observed that subjects with early RA or at risk of developing RA are more likely to produce IL-6 to no epitope or to citrullinated aggrecan alone, and consequently the T lymphocyte response in those subjects may be characterized essentially by the production of IL-6. Accordingly, in specific embodiments, the methods of the present invention may further comprise identifying that the subject produces IL-6 prior to eliciting the antigen-specific tolerogenic response.
In order to identify whether a subject is making the T lymphocyte response, the methods of the invention may employ assays for detection or assessment of released cytokine(s). The cytokine(s) may be assayed using any suitable technique. Standard assays for determining cytokine levels include functional activity protocols such as but not limited to: (a) cytokine-induced proliferation of indicator cell lines; (b) cytokine-induced apoptosis; (c) cytokine-induced protection from viral infection; and (d) cytokine-induced cytokine production. Such assays are well known to the skilled person and can be found, for example, at “Cytokine Bioassays” (www.ebioscience.com/ebioscience/appls/BAC.htm), which is incorporated herein by reference. Alternatively, the cytokine levels may be determined using conventional cytokine release assays, intracellular cytokine secretion assays, detection of activation of signal transduction pathways downstream of the T cell receptor and microarray detection of mRNA for species of proteins that indicate a T cell response such as cytokine mRNA, ELISAs, bead arrays or multiplex assays, which are well known to the skilled person. Non-limiting bioassays for IL-6, IFN-γ, TNF and IL-10 are disclosed in the Examples.
The present inventors have also determined that the T lymphocyte response in subjects with early RA or at risk of developing RA is produced at least in part by CD4+ CD28− T lymphocytes and/or CD4+ CD28+ T lymphocytes. Such T lymphocytes can be routinely detected using standard immunoassays including, for example, flow cytometry (e.g., using a fluorescence activated cell sorter (FACS)), immunohistochemistry, immunomagnetic separation and the like. Non-limiting immunoassays of this type are disclosed in the Examples. The T lymphocytes for analysis may be obtained from any suitable biological sample from the subject, representative samples of which include blood (e.g., peripheral blood) and synovial fluid.
3.2 Immune Modulators for Producing an Aggrecan-Specific Tolerogenic ResponseThe antigen-specific tolerogenic response may be elicited using any suitable strategy, illustrative examples of which include: (1) increasing the number of tolerogenic antigen-presenting cells in the subject, which present a peptide (e.g., an autoantigen) corresponding to a portion of the aggrecan polypeptide (also referred to herein as “aggrecan-specific tolerogenic antigen-presenting cells” or “agg-tolAPC”), wherein the portion is associated with the pro-inflammatory or autoreactive T lymphocyte response to the aggrecan polypeptide (e.g., a cit-agg polypeptide); (2) inducing anergy or apoptosis in autoreactive effector lymphocytes in the subject, which are reactive against the aggrecan polypeptide (e.g., a cit-agg polypeptide) or portion thereof; and (3) increasing the number of regulatory or suppressor T lymphocytes in the subject, which suppress or otherwise reduce the autoreactive T lymphocyte response.
Aggrecan-specific tolAPC may be produced by contacting antigen-presenting cells (e.g., dendritic cells, macrophages, Langerhans cells etc.) with an inhibitor of the NF-κB pathway in an amount sufficient to inhibit the NF-κB pathway of the antigen-presenting cells and with an antigenic molecule selected from an antigen that corresponds in whole, or in part, to an aggrecan polypeptide (e.g., a cit-aggrecan polypeptide) or a nucleic acid molecule from which the antigen is expressible, in an amount sufficient for the antigen-presenting cells to present the antigen or a processed form thereof on their surface. Non-limiting methods for producing tolerogenic APC in vivo and/or in vitro are described in U.S. Pat. Appl. Pubs. 20030153518, 20060182726, 20100151000 and European Patent Application 1462111. In other embodiments, aggrecan-specific tolAPC are produced by contacting the antigen-presenting cells with an mTOR inhibitor or a Syk pathway inhibitor in an amount sufficient to inhibit mTOR or the Syk pathway of the antigen-presenting cells, together with an antigenic molecule as described above.
Antigen-presenting cells include both professional and facultative types of antigen-presenting cells. Professional antigen-presenting cells include, but are not limited to, macrophages, monocytes, B lymphocytes, cells of myeloid lineage, including monocytic-granulocytic-DC precursors, marginal zone Küpffer cells, microglia, T cells, Langerhans cells and dendritic cells including interdigitating dendritic cells and follicular dendritic cells. Examples of facultative antigen-presenting cells include but are not limited to activated T cells, astrocytes, follicular cells, endothelium and fibroblasts. In some embodiments, the antigen-presenting cell is selected from monocytes, macrophages, B-lymphocytes, cells of myeloid lineage, dendritic cells or Langerhans cells.
3.2.1 Aggrecan AntigenThe aggrecan antigen may comprise, consist or consist essentially of an amino acid sequence corresponding to a putatively full-length aggrecan polypeptide (e.g., as set forth in SEQ ID NO: 2 or 4, including citrullinated forms thereof). Alternatively, the antigen may comprise, consist or consist essentially of an amino acid sequence corresponding to a mature aggrecan polypeptide (e.g., as set forth in SEQ ID NO: 37 or 39, including citrullinated forms thereof) or to a domain thereof such as but not limited to the G1 domain (e.g., as set forth in SEQ ID NO: 41, including citrullinated forms thereof), the G2 domain (e.g., as set forth in SEQ ID NO: 43, including citrullinated forms thereof) or the G3 domain (e.g., as set forth in SEQ ID NO: 43, including citrullinated forms thereof). In other embodiments, the antigen may comprise, consist or consist essentially of an amino acid sequence corresponding to a T cell epitope (e.g., an autoantigen) of an aggrecan polypeptide (e.g., as set forth in any one of SEQ ID NO: 5-35, including citrullinated forms thereof). In specific embodiments, the antigen comprises, consists or consists essentially of an amino acid sequence as set forth in any one of SEQ ID NO: 32-35. In non-limiting examples, the antigen is HLA DR restricted and the subject is suitably positive for an HLA DR allele. In these examples, HLA DR-binding peptides (e.g., HLA-DRB-binding peptides) may be predicted using any suitable approach including the use of predictive algorithms as described for example by James et al. (2009, J Immunol 183:3249-3258; and 2010, Arthritis Rheum. 62(10):2909-2918), Knapp et al. (2011, BMC Bioinformatics 12:241), Honeyman et al. (1997, Ann. Med. 29:401-404) and Hu et al. (2010, Nucleic Acids Research 38:Web Server issue W474-W479).
In some embodiments, the antigen is in the form of one or more peptides corresponding in whole or in part to an aggrecan polypeptide, including citrullinated forms thereof. Usually, such peptides are at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30 amino acid residues in length and suitably no more than about 500, 200, 100, 80, 60, 50, 40 amino acid residues in length. In some embodiments in which two or more peptides are used, the peptides can be in the form of a plurality of contiguous overlapping peptides whose sequences span at least a portion of an aggrecan polypeptide, including citrullinated forms thereof. Suitably, the peptide sequences are derived from at least about 30, 40, 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% of the sequence corresponding to the aggrecan polypeptide, including citrullinated forms thereof. In some embodiments, each peptide of the plurality of contiguous overlapping peptide fragments can be 30-90 amino acids in length, e.g., 30, 35, 40, 45, 50, 55, 60, 65, 70, 73, 75, 80, 81, 85, 86 and 90 amino acids in length. In various embodiments, the amino acid sequences of contiguous overlapping peptide fragments in the plurality overlap by about 10 to about 15 amino acids, e.g., 10, 11, 12, 13, 14 and 15 amino acids. Suitably, the length of the peptides is selected for presentation to cytolytic T lymphocytes (e.g., peptides of about 8 to about 10 amino acids in length), or for presentation to T helper lymphocytes (e.g., peptides of about 12 to about 20 amino acids in length). Exemplary methods for producing such peptide antigens are described, for example, by Astori et al. (2000, J. Immunol. 165:3497-3505; and references cited therein) and in U.S. Pat. Appl. Pub. No. 2004/0241178. If desired, the aggrecan antigen may be modified, for example, by lipid modification to modify its physico-chemical properties. Non-limiting examples of overlapping peptides spanning substantially the entire length of an aggrecan polypeptide, are set forth in SEQ ID NO: 49-531.
The aggrecan antigen(s) may be isolated from a natural source (e.g., from the subject). Alternatively, they may be prepared in recombinant form using standard protocols as for example described in: Sambrook, et al., MOLECULAR CLONING. A LABORATORY MANUAL (Cold Spring Harbor Press, 1989), in particular Sections 16 and 17; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (John Wiley & Sons, Inc. 1994-1998), in particular Chapters 10 and 16; and Coligan et al., CURRENT PROTOCOLS IN PROTEIN SCIENCE (John Wiley & Sons, Inc. 1995-1997), in particular Chapters 1, 5 and 6. In representative examples, the aggrecan antigen(s) may be prepared by a procedure including the steps of (a) providing an expression vector from which a nucleotide sequence encoding the aggrecan antigen is expressible; (b) introducing the vector into a suitable host cell; (c) culturing the host cell to express recombinant polypeptide from the vector; and (d) isolating the recombinant antigen. In specific embodiments, the aggrecan antigen(s) correspond to the sequence of an aggrecan polypeptide, whether citrullinated or not, which is produced by the species of animal to which the subject belongs.
Alternatively, the aggrecan antigen(s) is (are) synthesized using solution synthesis or solid phase synthesis as described, for example, by Atherton and Sheppard (Solid Phase Peptide Synthesis: A Practical Approach, IRL Press at Oxford University Press, Oxford, England, 1989) or by Roberge et al. (1995, Science 269:202).
In some embodiments, the aggrecan antigen comprises at least one citrulline residue. Citrullinated antigens can, for example, be obtained from natural, recombinant or synthetic forms of aggrecan antigen, through the action of peptidylarginine deiminase (PAD); they can also be obtained by peptide synthesis, by directly incorporating one or more citrulline residues into a synthesized aggrecan peptide. These methods are well known to the skilled practitioner. Illustrative methods of using PAD enzymes for making citrullinated antigens are described in U.S. Pat. Appl. Pub. No. 2011/0028399. Exemplary methods of synthesizing citrullinated antigens are described in Perez et al., 2006. Chem Biol Drug Des 68:194-200)
In other embodiments, the aggrecan antigen(s) are provided in nucleic acid form, generally by operably linking a nucleotide sequence that encodes the antigen(s) to a promoter, which may be constitutive or inducible, and which is operable in antigen-presenting cells of interest, to form a nucleic acid construct. Delivery of the nucleic acid constructs into antigen-presenting cells or their precursors may be achieved either by directly exposing a patient to the nucleic acid construct or by first transforming antigen-presenting cells or their precursors with the nucleic acid construct in vitro, and then transplanting the transformed antigen-presenting cells or precursors into the patient.
The nucleic acid construct may be introduced into the antigen-presenting cell by any suitable means including for example by contacting the antigen-presenting cell with the nucleic acid construct, electroporation, transformation, transduction, conjugation or triparental mating, transfection, infection membrane fusion with cationic lipids, high-velocity bombardment with DNA-coated microprojectiles, incubation with calcium phosphate-DNA precipitate, direct microinjection into single cells, and the like. Other methods also are available and are known to those skilled in the art. In specific embodiments, the nucleic acid constructs are introduced by means of cationic lipids, e.g., liposomes. Such liposomes are commercially available (e.g., Lipofectin®, Lipofectamine™, and the like, supplied by Life Technologies, Gibco BRL, Gaithersburg, Md.).
The antigen-encoding nucleotide sequence of the construct may comprise a naturally occurring sequence or a variant thereof, which has been engineered using recombinant techniques. In one example of a variant, the antigen-encoding nucleotide sequence is codon optimized to permit enhanced expression of the antigen in an antigen-presenting cell of interest. Illustrative methods for codon optimization are described for example in U.S. Pat. No. 5,795,737, WO 99/02694 and WO 00/42215.
The delivery of aggrecan antigen to an antigen-presenting cell or its precursor can be enhanced by methods known to practitioners in the art. For example, several different strategies have been developed for delivery of exogenous antigen to the endogenous processing pathway of antigen-presenting cells, especially dendritic cells. These methods include insertion of antigen into pH-sensitive liposomes (Zhou and Huang, 1994. Immunomethods 4:229-235), osmotic lysis of pinosomes after pinocytic uptake of soluble antigen (Moore et al., 1988. Cell 54:777-785), coupling of antigens to potent adjuvants (Aichele et al., 1990. J. Exp. Med. 171:1815-1820; Gao et al., 1991. J. Immunol. 147:3268-3273; Schulz et al., 1991. Proc. Natl. Acad. Sci. USA 88:991-993; Kuzu et al., 1993. Euro. J. Immunol. 23:1397-1400; and Jondal et al., 1996. Immunity 5:295-302), exosomes (Zitvogel et al., 1998. Nat Med. 4:594-600; 2002, Nat Rev Immunol. 2:569-79), and apoptotic cell delivery of antigen (Albert et al., 1998. Nature 392:86-89; Albert et al., 1998, Nature Med. 4:1321-1324; and in International Publications WO 99/42564 and WO 01/85207). Recombinant bacteria (e.g., E. coli) or transfected host mammalian cells may be pulsed onto dendritic cells (as particulate antigen, or apoptotic bodies respectively) for antigen delivery. Recombinant chimeric virus-like particles (VLPs) have also been used as vehicles for delivery of exogenous heterologous antigen to the MHC class I processing pathway of a dendritic cell line (Bachmann et al., 1996. Eur. J. Immunol. 26(11):2595-2600).
Alternatively, or in addition, an aggrecan antigen may be linked to, or otherwise associated with, a cytolysin to enhance the transfer of the antigen into the cytosol of an antigen-presenting cell of the invention for delivery to the MHC class I pathway. Exemplary cytolysins include saponin compounds such as saponin-containing Immune Stimulating Complexes (ISCOMs) (see e.g., Cox and Coulter, 1997. Vaccine 15(3):248-256 and U.S. Pat. No. 6,352,697), phospholipases (see, e.g., Camilli et al., 1991. J. Exp. Med. 173:751-754), pore-forming toxins (e.g., an alpha-toxin), natural cytolysins of gram-positive bacteria, such as listeriolysin O (LLO, e.g., Mengaud et al., 1988. Infect. Immun. 56:766-772 and Portnoy et al., 1992. Infect. Immun. 60:2710-2717), streptolysin O (SLO, e.g., Palmer et al., 1998. Biochemistry 37(8):2378-2383) and perfringolysin O (PFO, e.g., Rossjohn et al., Cell 89(5), 685-692). Where the antigen-presenting cell is phagosomal, acid activated cytolysins may be advantageously used. For example, listeriolysin exhibits greater pore-forming ability at mildly acidic pH (the pH conditions within the phagosome), thereby facilitating delivery of vacuole (including phagosome and endosome) contents to the cytoplasm (see, e.g., Portnoy et al., 1992. Infect. Immun. 60:2710-2717).
The cytolysin may be provided together with an aggrecan antigen in the form of a single composition or may be provided as a separate composition, for contacting the antigen-presenting cells. In one embodiment, the cytolysin is fused or otherwise linked to the antigen, wherein the fusion or linkage permits the delivery of the antigen to the cytosol of the target cell. In another embodiment, the cytolysin and antigen are provided in the form of a delivery vehicle such as, but not limited to, a liposome or a microbial delivery vehicle selected from virus, bacterium, or yeast. Suitably, when the delivery vehicle is a microbial delivery vehicle, the delivery vehicle is non-virulent. In a preferred embodiment of this type, the delivery vehicle is a non-virulent bacterium, as for example described by Portnoy et al. in U.S. Pat. No. 6,287,556, comprising a first polynucleotide encoding a non-secreted functional cytolysin operably linked to a regulatory polynucleotide which expresses the cytolysin in the bacterium, and a second polynucleotide encoding one or more pre-selected antigens. Non-secreted cytolysins may be provided by various mechanisms, e.g., absence of a functional signal sequence, a secretion incompetent microbe, such as microbes having genetic lesions (e.g., a functional signal sequence mutation), or poisoned microbes, etc. A wide variety of nonvirulent, non-pathogenic bacteria may be used; preferred microbes are relatively well-characterized strains, particularly laboratory strains of E. coli, such as MC4100, MC1061, DH5α, etc. Other bacteria that can be engineered for the invention include well-characterized, nonvirulent, non-pathogenic strains of Listeria monocytogenes, Shigella flexneri, mycobacterium, Salmonella, Bacillus subtilis, etc. In a particular embodiment, the bacteria are attenuated to be non-replicative, non-integrative into the host cell genome, and/or non-motile inter- or intra-cellularly.
The delivery vehicles described above can be used to deliver one or more aggrecan antigens to virtually any antigen-presenting cell capable of endocytosis of the subject vehicle, including phagocytic and non-phagocytic antigen-presenting cells. In embodiments when the delivery vehicle is a microbe, the subject methods generally require microbial uptake by the target cell and subsequent lysis within the antigen-presenting cell vacuole (including phagosomes and endosomes).
3.2.2 Altered Peptide LigandsIn some embodiments, an altered peptide ligand (APL) is used for stimulating aggrecan-specific tolerogenesis. The APL suitably comprises, consists or consists essentially of an amino acid sequence corresponding to a portion (e.g., an autoantigen) of an aggrecan polypeptide (e.g., a cit-aggrecan polypeptide), which is suitably associated with rheumatoid arthritis (RA), and which binds to an antigen receptor (e.g., T cell receptor) on pro-inflammatory or autoreactive T lymphocytes. The APL amino acid sequence is generally distinguished from the amino acid sequence of the aggrecan polypeptide portion by at least one amino acid substitution, deletion or addition, and suitably interferes with normal signaling through the antigen receptor.
APL can be designed based on natural aggrecan peptide epitopes identified using any method known in the art. For example, methods involving isolating and assaying MHC molecules from antigen presenting cells can be used to identify peptides bound to the MHC molecules (Chicz and Urban, 1994. Immunol. Today 1-5:155-160. Bacteriophage “phage display” libraries can also be constructed. Using the “phage method” (Scott and Smith, 1990. Science 249:386-390; Cwirla et al., 1990. Proc. Natl. Acad. Sci. USA 87:6378-6382; Devlin et al., 1990. Science 249:404-406), very large libraries can be constructed (106-108 chemical entities). Other methods to identify peptide epitopes which can be used involve primarily chemical methods, of which the Geysen method (Geysen et al., 1986. Molecular Immunology 23:709-715; Geysen et al., 1987. J. Immunologic Method 102:259-274; and the method of Fodor et al., 1991. Science 251:767-773) are examples. Furka et al., 1988. 14th International Congress of Biochemistry, Volume 5. Abstract FR:013; Furka, 1991. Int. J. Peptide Protein Res. 37:487-493). Houghton (U.S. Pat. No. 4,631,211) and Rutter et al. (U.S. Pat. No. 5,101,175) describe methods to produce a mixture of peptides that can be tested as agonists or antagonists. Other methods which can be employed involve use of synthetic libraries (Needels et al., 1993. Proc. Natl. Acad. Sci. USA 90:10700-4; Ohlmeyer et al., 1993. Proc. Natl. Acad. Sci. USA 90:10922-10926; Lam et al., International Patent Publication No. WO 92/00252, each of which is incorporated herein by reference in its entirety), and the like can be used to screen for receptor ligands. Techniques based on cDNA subtraction or differential display have been described amply in the literature and can also be used see, for example, Hedrick et al., 1984. Nature 308:149; and Lian and Pardee, 1992. Science 257:967. The expressed sequence tag (EST) approach is a valuable tool for gene discovery (Adams et al., 1991. Science 252:1651), as are Northern blotting, RNase protection, and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis (Alwine et al., 1977. Proc. Natl. Acad. Sci. USA 74:5350; Zinn et al., 1983. Cell 34:865; Veres et al., 1987. Science 237:415). Another technique which can be used is the “pepscan” technique (Van der Zee, 1989. Eur. J. Immunol. 19:43-47) in which several dozens of peptides are simultaneously synthesized on polyethylene rods arrayed in a 96-well microtiter plate pattern, similar to an indexed library in that the position of each pin defines the synthesis history on it. Peptides are then chemically cleaved from the solid support and supplied to irradiated syngeneic thymocytes for antigen presentation. A cloned CTL line is then tested for reactivity in a proliferation assay monitored by 3H-thymidine incorporation.
SPHERE is described in WO 97/35035. This approach utilizes combinatorial peptide libraries synthesized on polystyrene beads wherein each bead contains a pure population of a unique peptide that can be chemically released from the beads in discrete aliquots. Released peptide from pooled bead arrays are screened using methods to detect T cell activation, including, for example, .sup.3H-thymidine incorporation (for CD4+ or CD8+ T cells), 51Cr-release assay (for CTLs) or IL-2 production (for CD4+ T cells) to identify peptide pools capable of activating a T cell of interest. By utilizing an iterative peptide pool/releasing strategy, it is possible to screen more than 107 peptides in just a few days. Analysis of residual peptide on the corresponding positive beads (>100 pmoles) allows rapid and unambiguous identification of the epitope sequence.
A brief overview of an assay to identify aggrecan peptides binding to CTLs is as follows: roughly speaking, ten 96-well plates with 1000 beads per well will accommodate 106 beads; ten 96-well plates with 100 beads per well will accommodate 105 beads. In order to minimize both the number of CTL cells required per screen and the amount of manual manipulations, the eluted peptides can be further pooled to yield wells with any desired complexity. For example, based on experiments with soluble libraries, it is possible to screen 107 peptides in 96-well plates (10,000 peptides per well) with as few as 2×106 CTL cells. After cleaving a percentage of the peptides from the beads, incubating them with gamma-irradiated foster APCs and the cloned CTL line(s), positive wells determined by 3H-thymidine incorporation are further examined. Alternatively, as pointed out above, cytokine production or cytolytic 51Cr-release assays may be used. Coulie et al., 1992. Int. J. Cancer 50:289-291. Beads from each positive well will be separated and assayed individually as before, utilizing an additional percentage of the peptide from each bead. Positive individual beads will then be decoded, identifying the reactive amino acid sequence. Analysis of all positives will give a partial profile of conservatively substituted epitopes that stimulate the CTL clone tested. At this point, the peptide can be resynthesized and retested. Also, a second library (of minimal complexity) can be synthesized with representations of all conservative substitutions in order to enumerate the complete spectrum of derivatives tolerated by a particular CTL. By screening multiple CTLs (of the same MHC restriction) simultaneously, the search for crossreacting epitopes is greatly facilitated.
The described method for the identification of CD8+ MHC Class I-restricted CTL epitopes can be applied to the identification of CD4+ MHC Class II-restricted CD4+ T cell epitopes. In this case, MHC Class II allele-specific libraries are synthesized such that haplotype-specific anchor residues are represented at the appropriate positions. MHC Class II agretopic motifs have been identified for the common alleles (see, for example, Rammensee, 1995. Curr. Opin. Immunol. 7:85-96; Altuvia et al., 1994. Mol. Immunol. 24:375-379; Reay et al., 1994. J. Immunol. 152:3946-3957; Verreck et al., 1994. Eur. J. Immunol. 24:375-379; Sinigaglia and Hammer, 1994. Curr. Opin. Immunol. 6:52-56; Rotzschke and Falk, 1994. Curr. Opin. Immunol. 6:45-51). The overall length of the peptides will generally be 12-20 amino acid residues, and previously described methods may be employed to limit library complexity. Aggrecan APLs may be synthesized using solution synthesis or solid phase synthesis as described, for example, by Atherton and Sheppard (1989, supra) or by Roberge et al. (1995, supra).
In specific embodiments, the APL has at least one activity selected from: (i) antagonizing the response of the T lymphocytes to the aggrecan polypeptide (e.g., a cit-aggrecan polypeptide), (ii) inducing anergy in aggrecan- or citrullinated aggrecan-specific T lymphocytes, (iii) inducing apoptosis in aggrecan- or citrullinated aggrecan-specific T lymphocytes, (iv) stimulating or inducing a aggrecan- or citrullinated aggrecan-specific Th2 immune response, (v) suppressing development of a aggrecan- or citrullinated aggrecan-specific Th1 immune response including suppressing the production of pro-inflammatory cytokines, (vi) stimulating activation of aggrecan- or citrullinated aggrecan-specific regulatory lymphocytes (e.g., T regulatory lymphocytes (Treg)), or (vii) preventing or inhibiting the activation of aggrecan- or citrullinated aggrecan-specific antigen-presenting cells by an inflammatory stimulus.
3.2.3 NF-κB InhibitorsThe NF-κB inhibitor includes any molecule or compound that reduces the level or functional activity of NF-κB in immune cells, especially antigen-presenting cells. NF-κB inhibitors can take various forms, non-limiting examples of which include small molecules, nucleic acids, peptides, polypeptides, peptidomimetics etc.
In some embodiments, the NF-κB inhibitor decreases the level or functional activity of a member of the NF-κB pathway, which is suitably selected from BTK, LYN, BCR Igα, BCR Igβ, Syk, Blnk, PLCγ2, PKCβ, DAG, CARMA1, BCL10, MALT1, PI3K, PIP3, AKT, p38 MAPK, ERK, COT, IKKα, IKKβ, IKKγ, NIK, RelA/p65, P105/p50, c-Rel, RelB, p52, NIK, Leu13, CD81, CD19, CD21 and its ligands in the complement and coagulation cascade, TRAF6, ubiquitin ligase, Tab2, TAK1, NEMO, NOD2, RIP2, Lck, fyn, Zap70, LAT, GRB2, SOS, CD3 zeta, Slp-76, GADS, ITK, PLCγ1, PKCθ, ICOS, CD28, SHP2, SAP, SLAM and 2B4. In illustrative examples of this type, the NF-κB inhibitor decreases the level or functional activity of any one or more of RelA/p65, P105/p50, c-Rel, RelB or p52. Suitably, in these embodiments, the NF-κB inhibitor blocks, inhibits or otherwise antagonizes at least one function or activity of the member. In other embodiments, the NF-κB inhibitor increases the level or functional activity of a member of the NF-κB pathway, which is suitably selected from SHP1, SHIP, PIR-B, CD22, CD72, FcgRIIB, IκB, P100, CTLA4, PD-1, Cbl, KIR3DL1, KIR3DL2, KIR2DL and Csk. In these embodiments, the NF-κB inhibitor increases, stimulates or otherwise agonizes at least one function or activity of the member.
A plethora of NF-κB inhibitors is described in the literature and representative examples are listed in the following tables:
In specific embodiments, the NF-κB inhibitor is an inhibitor of IκB phosphorylation, such as BAY 11-7082 and BAY 11-7085 (BioMol, Plymouth Meeting, Pa.), curcumin and curcumin derivatives or analogs. Numerous curcumin derivatives and analogs are known in the art and may be used in the present invention (see, e.g., WO 2007/051314; US 2006/0276536). Such derivatives may have increased solubility or potency. Examples of curcumin derivatives include dimethoxy curcumin (Jeong et al., 2009. J. Clin. Biochem. Nutr. 44:79-84), 3,5-bis(2-fluorobenzylidene)-4-piperidone (EF24) (U.S. Pat. Appl. Pub. 20110059157), bis(arylmethylidene)acetone (WO 2007/000998), desmethoxy curcumin and bisdesmethoxy curcumin (WO 2006/117077). Other curcumin analogs that may be used include dihydrocurcumin, tetrahydrocurcumin, hexahydrocurcumin, dihydroxytetrahydrocurcumin, Yakuchinone A and Yakuchinone B, and their salts, oxidants, reductants, glycosides and esters thereof (U.S. Pat. Appl. Pub. 20030147979; U.S. Pat. No. 5,891,924). Further examples of curcumin analogs include but are not limited to (a) ferulic acid, (e.g., 4-hydroxy-3-methoxycinnamic acid; 3,4-methylenedioxy cinnamic acid; and 3,4-dimethoxycinnamic acid); (b) aromatic ketones (e.g., 4-(4-hydroxy-3-methoxyphenyl)-3-buten-2-one; zingerone; 4-(3,4-methylenedioxyphenyl)-2-butanone; 4-(p-hydroxyphenyl)-3-buten-2-one; 4-hydroxyvalerophenone; 4-liydroxybenzylactone; 4-hydroxybenzophenone; 1,5-bis(4-dimethylaminophenyl)-1,4-pentadien-3-one); (c) aromatic diketones (e.g., 6-hydroxydibenzoylmethane) (d) caffeic acid compounds (e.g., 3,4-dihydroxycinnamic acid); (e) cinnamic acid; (i) aromatic carboxylic acids (e.g., 3,4-dihydroxyhydrocinnainic acid; 2-hydroxycinnamic acid; 3-hydroxycinnamic acid and 4-liydroxycinnamic acid); (g) aromatic ketocarboxylic acids (e.g., 4-hydroxyphenylpyruvic acid); and (h) aromatic alcohols (e.g., 4-hydroxyphenethyl alcohol). These analogs and other representative analogs that can be used in the present invention are further described in WO 95/18606 and WO 01/040188. Other curcumin derivatives and analogs, including dimers, dextran and dendrimer conjugates, may also be used (see, e.g., U.S. Pat. Appl. Pub. 20100240905; Shi, W., et al., 2007. Org. Lett. 9(26):5461-5464).
The curcumin or curcumin derivative or analog may be provided as a conjugate such as a prodrug. Examples of curcumin prodrugs are known (see, e.g., Lu, P., et al., 2005. J Huazhong Univ Sci Technolog Med. Sci. 25(6):668-670, 678, Kapoor, N., et al. 2007. Cancer Lett. 248(2):245-250), and methods of making prodrugs are known (see, e.g., WO 2006/076734; U.S. Pat. No. 5,952,294; Balant, L. P., et al., 1990. Eur. J. Drug Metab. Pharmacokinet. 15:143-153; Bundgaard, H., et al., 1991. Drugs of the Future 16:443-458). See also U.S. Published Patent Application U.S. 2007/0270464.
Desirably, the NF-κB inhibitor is non-toxic to the host with minimal or negligible side effects. Suitably, the inhibitor of NF-κB blocks the alternate NF-κB pathway, or both the classical and the alternate NF-κB pathways.
In some embodiments, the NF-κB inhibitor is in nucleic acid form, generally by operable linkage of a nucleotide sequence that encodes the inhibitor to a promoter, which may be constitutive or inducible, and which is operable in antigen-presenting cells of interest, to form a nucleic acid construct. Delivery of the nucleic acid constructs into antigen-presenting cells or their precursors may be achieved either by directly exposing a patient to the nucleic acid construct or by first transforming antigen-presenting cells or their precursors with the nucleic acid construct in vitro, and then transplanting the transformed antigen-presenting cells or precursors into the patient.
Considerations for introducing nucleic acid constructs into antigen-presenting cells and for producing variant nucleotide sequences as discussed in Section 3.2.1 for antigen-encoding nucleic acid constructs apply equally to nucleic acid constructs from which NF-κB inhibitor peptides/polypeptides are expressible.
3.2.4 mTOR InhibitorsInhibition of the mTOR pathway is known to stimulate tolerogenicity in antigen-presenting cells (e.g., dendritic cells, B cells etc.) as described for example by Delgoffe and Powell (2009. Immunology 127(4):459-465) and Fischer et al. (2009, Handb Exp Pharmacol. 188:215-232). Accordingly, the present invention contemplates the use of inhibitors of the mTOR pathway, together with an antigenic molecule selected from an antigen that corresponds in whole, or in part, to an aggrecan polypeptide (e.g., a cit-aggrecan polypeptide) or a nucleic acid molecule from which the antigen is expressible, for suppressing the immune response to an aggrecan polypeptide, in the treatment or prevention of joint damage in a subject.
As used herein, mTOR inhibitors include any molecule or compound that reduces the level or functional activity of mTOR in immune cells, especially antigen-presenting cells. mTOR inhibitors can take various forms, non-limiting examples of which include small molecules, nucleic acids, peptides, polypeptides, peptidomimetics etc.
In some embodiments, the mTOR inhibitor is selected from rapamycin, which is a known macrolide antibiotic produced by Streptomyces hygroscopicus, and rapamycin derivatives, e.g., rapamycin substituted in position 40 and/or 16 and/or 32, for example a compound of formula (I):
wherein R1 is CH3 or C3-6alkynyl,
R2 is H, —CH2—CH2—OH, or —CH2—CH2—O—CH2—CH3.
3-hydroxy-2-(hydroxymethyl)-2-methyl-propanoyl or tetrazolyl, e.g. tetrazol-1-yl, and X is ═O, (H, H) or (H, OH), provided that R2 is other than H when X is ═O and R1 is CH3.
When R2 in a compound of formula I is —CH2—CH2—OH, a compound of formula I includes a physiologically hydrolysable ether thereof, for instance —CH2—CH2—O—C1-8)alkyl.
Representative examples of compounds of formula I include e.g. 40-O-(2-hydroxyl)ethyl-rapamycin (also known as everolimus), 32-deoxorapamycin, 16-O-substituted rapamycins such as 16-pent-2-ynyloxy-32-deoxorapamycin, 16-pent-2-ynyloxy-32 (S or R)-dihydro-rapamycin, 16-pent-2-ynyloxy-32 (S or R)-dihydro-40-0-(2-hydroxyethyl)-rapamycin, 40-[3-hydroxy-2-(hydroxy-methyl)-2-methylpropanoate]-rapamycin (also known as CCI779), 40-epi-(tetrazolyl)-rapamycin (also known as ABT578), or 40-O-ethoxyethyl-rapamycin (also known as biolimus 9).
mTOR inhibitors also include the so-called rapalogs, e.g., as disclosed in WO9802441, WO0114387 and WO0364383 (which are hereby incorporated by reference in their entirety) such as AP23573, e.g., 40-O-(dimethylphosphinoyl)-rapamycin, compounds as disclosed disclosed in WO2005047295 in Example 1, also designated as biolimus A9 and compounds disclosed under the name TAFA-93. Other mTOR inhibitors are e.g., disclosed in WO2004101583, WO9205179, WO9402136, WO9402385, WO9613273, which are hereby incorporated by reference in their entirety.
In some embodiments, mTOR inhibitors include rapamycin, a compound of formula I, e.g., and including a rapalog, TAFA-93, more preferably rapamycin, a compound of formula I or a rapalog,
In specific embodiments, the mTOR inhibitor is 40-O-(2-hydroxyethyl)-rapamycin disclosed in Example 8 in WO9409010.
In other embodiments, the mTOR inhibitor is 32-deoxorapamycin or 16-pent-2-ynyloxy-32 (S)-dihydro-rapamycin as disclosed in WO9641807, e.g., or a compound as disclosed in WO9516691.
Exemplary mTOR inhibitors include: rapamycin, and/or 40-O-(2-hydroxyethyl)-rapamycin, and/or 32-deoxorapamycin, and/or 16-pent-2-ynyloxy-32-deoxorapamycin, and/or 16-pent-2-ynyloxy-32 (S or R)-dihydro-rapamycin, and/or 16-pent-2-ynyloxy-32 (S or R)-dihydro-40-0-(2-hydroxyethyl)-rapamycin, and/or 40-[3-hydroxy-2-(hydroxy-methyl)-2-methylpropanoate]-rapamycin (also known as CCI779) and/or 40-epi-(tetrazolyl)-rapamycin (also known as ABT578), and/or AP23573, and/or biolimus A9, e.g., and/or compounds disclosed under the name TAFA-93, such as 40-O-(2-hydroxyethyl)-rapamycin, and/or 32-deoxorapamycin, and/or CCI779, and/or ABT578, and/or AP23573.
In other embodiments, mTOR inhibitors are selected from pyrazolopyrimidine derivatives, including but not limited to compounds having a structure of the following formula (II) and formula (III).
In some embodiments of the compounds of formulae (II) or (III), R1, R3, and R4 are independently hydrogen, halogen, CN, —CF3, —OH, —NH2, —SO2, —COOH, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. In some embodiments, at least one of R3 or R4 is hydrogen. In some embodiments, R1, R3, and R4 are independently hydrogen or substituted or unsubstituted C1-C10 alkyl (e.g. C1-C5 alkyl or C1-C3 alkyl). R′, R3, and R4 may also independently be hydrogen or unsubstituted C1-C10 alkyl (e.g. C1-C5 alkyl or C1-C3 alkyl).
In some embodiments of formulae (II) or (III), R1, R3, and R4 are independently hydrogen, halogen, —CN, —CF3, —OH, —NH2, —SO2, —COOH, R7-substituted or unsubstituted alkyl, R7-substituted or unsubstituted heteroalkyl, R7-substituted or unsubstituted cycloalkyl, R7-substituted or unsubstituted heterocycloalkyl, R7-substituted or unsubstituted aryl, or R7-substituted or unsubstituted heteroaryl. R7 is independently oxo, halogen, —CN, —CF3, —OH, —NH2, —SO2, —COOH, R8-substituted or unsubstituted alkyl, R8-substituted or unsubstituted heteroalkyl, R8-substituted or unsubstituted cycloalkyl, R8-substituted or unsubstituted heterocycloalkyl, R8-substituted or unsubstituted aryl, or R8-substituted or unsubstituted heteroaryl. R8 is independently halogen, oxo, —CN, —CF3, —OH, —NH2, —SO2, —COOH, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl or unsubstituted heteroaryl. In some embodiments, R1 is substituted or unsubstituted alkyl or substituted or unsubstituted heterocycloalkyl (e.g. morpholino). In some embodiments, R1 is substituted with —C(O)R8A, wherein R8A is unsubstituted alkyl.
In some embodiments of formula (II), R2 is independently hydrogen, halogen, —CN, —CF3, —OR5, —NH2, —SO2, —COOH, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. In some embodiments, R2 is independently hydrogen, —OR5, —CN, —NH2, —SH, —CN, —CF3, NO2, or substituted or unsubstituted alkyl. R5 is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. In some embodiments, R5 is hydrogen or substituted or unsubstituted alkyl (e.g. unsubstituted C1-C5 alkyl).
R2 may independently be hydrogen, halogen, —OR5, —CN, —NH2, —SH, —CN, —CF3, —NO2, R9-substituted or unsubstituted alkyl, R9-substituted or unsubstituted heteroalkyl, R9-substituted or unsubstituted cycloalkyl, R9-substituted or unsubstituted heterocycloalkyl, R9-substituted or unsubstituted aryl, or R9-substituted or unsubstituted heteroaryl. R9 is independently halogen, oxo, —CN, —CF3, —OH, —NH2, —SO2, —COOH, R10-substituted or unsubstituted alkyl, R10-substituted or unsubstituted heteroalkyl, R10-substituted or unsubstituted cycloalkyl, R10-substituted or unsubstituted heterocycloalkyl, R10-substituted or unsubstituted aryl, or R10-substituted or unsubstituted heteroaryl. R10 is independently halogen, oxo, —CN, —CF3, —OH, —NH2, —SO2, —COOH, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl or unsubstituted heteroaryl. In other embodiments, R2 is —OR5. In some related embodiments, R5 is hydrogen or unsubstituted C1-C5 alkyl (e.g. hydrogen).
In some embodiments of formulae (II) or (III), R5 is independently hydrogen, R11-substituted or unsubstituted alkyl, R11-substituted or unsubstituted heteroalkyl, R11-substituted or unsubstituted cycloalkyl, R11-substituted or unsubstituted heterocycloalkyl, R11-substituted or unsubstituted aryl, or R11-substituted or unsubstituted heteroaryl. R11 is independently halogen, oxo, —CN, —CF3, —OH, —NH2, —SO2, —COOH, R12-substituted or unsubstituted alkyl, R12-substituted or unsubstituted heteroalkyl, R12-substituted or unsubstituted cycloalkyl, R12-substituted or unsubstituted heterocycloalkyl, R12-substituted or unsubstituted aryl, or R12-substituted or unsubstituted heteroaryl. R12 is independently halogen, oxo, —CN, —CF3, —OH, —NH2, —SO2, —COOH, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl or unsubstituted heteroaryl.
In some embodiments of formula (III), R6 is independently hydrogen, halogen, —CN, —CF3, —OR5, —NH2, —SO2, —COOH, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. R6 may also independently be hydrogen, —OR5, —CN, halogen, —NH2, —SH, —CN, —CF3, —NO2, halogen, or substituted or unsubstituted alkyl (e.g. unsubstituted C1-C5 alkyl). R5 is as defined above in the description of Formula (I). In some embodiments, R6 is independently hydrogen, —OR5, —CN, —NH2, —SH, —CN, —CF3, —NO2, R13-substituted or unsubstituted alkyl, R13-substituted or unsubstituted heteroalkyl, R13-substituted or unsubstituted cycloalkyl, R13-substituted or unsubstituted heterocycloalkyl, R13-substituted or unsubstituted aryl, or R13-substituted or unsubstituted heteroaryl. R13 is independently halogen, oxo, —CN, —CF3, —OH, —NH2, —SO2, —COOH, R14-substituted or unsubstituted alkyl, R13-substituted or unsubstituted heteroalkyl, R14-substituted or unsubstituted cycloalkyl, R14-substituted or unsubstituted heterocycloalkyl, R14-substituted or unsubstituted aryl, or R14-substituted or unsubstituted heteroaryl. R14 is independently halogen, oxo, —CN, —CF3, —OH, —NH2, —SO2, —COOH, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl or unsubstituted heteroaryl. R6 may also independently be hydrogen, —OR5, —CN, halogen, —NH2, —SH, —CN, —CF3, —NO2, halogen, or unsubstituted C1-C5 alkyl. In some embodiments, R6 is hydrogen.
In some embodiments of formulae (II) or (III), R3 and R4 are hydrogen. In some embodiments of formula (II), n is 1 or 2. In some related embodiments of formula (II), n is 1. In other related embodiments, R2 is —OR5 and n is 1. In still other related embodiments, R5 is hydrogen. In some embodiments of formula (III), z is an integer from 1 to 2. In some embodiments, z is 1.
In some embodiments, R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12, R13 and/or R14 are size-limited substituents. In some embodiments, R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12, R13 and/or R14 are C1-C10, C1-C5 alkyl or C1-C3 alkyl, for example methyl, ethyl, propyl, isopropyl, butyl and the like, optionally substituted as described herein. In some embodiments, R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12, R13 and/or R14 are 2-10 membered, 2-5 membered, or 2-3-membered heteroalkyl, optionally substituted as described herein. In some embodiments, R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12, R13 and/or R14 are C3-C10, C3-C8, C3-C6 or C3-C5 cycloalkyl, including but not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and the like, optionally substituted as described herein. In some embodiments, R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12, R13 and/or R14 are 3-membered, 4-membered, 5-membered, 6-membered, 7-membered, 8-membered, 9-membered or 10-membered heterocycloalkyl, including but not limited to aziridine, oxirane, thiirane, azetidine, oxetane, thietane, pyrrolidine, dihydrofuran, tetrahydropyran, dihydrothiophene, tetrahydrothiophene, piperidine, dihydropyran, tetrahydropyran, dihydrothiopyran, tetrahydrothiopyran, optionally substituted as described herein. In some embodiments, R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12, R13 and/or R14 are C6-C10 aryl, including but not limited to phenyl or naphthyl, optionally substituted as described herein. In some embodiments, R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12, R13 and/or R14 are 5-10-membered, 5-6-membered heteroaryl as described herein, optionally substituted as described herein.
In other embodiments, the biologically active agent (e.g. the compounds of formulae (II) or (III)) is selected from the following compounds:
Alternatively, or in addition, tolerogenicity in antigen-presenting cells (e.g., dendritic cells, B cells etc.) can be stimulated by inhibiting the Syk pathway, as described for example by Colonna et al. (2010. J Immunol. 185(3):1532-43), Matsubara et al. (2006. Am J Respir Cell Mol Biol. 34(4):426-433) and Nakashima et al. (2004. Eur J Pharmacol. 505(1-3):223-228). Thus, the present invention also contemplates the use of Syk inhibitors, in combination with an antigenic molecule selected from an antigen that corresponds in whole, or in part, to an aggrecan polypeptide (e.g., a cit-aggrecan polypeptide) or a nucleic acid molecule from which the antigen is expressible, for suppressing the immune response to an aggrecan polypeptide, and for treating or preventing joint damage in a subject.
As used herein, Syk pathway inhibitors include any molecule or compound that reduces signaling through the Syk pathway or that reduces the level or functional activity of Syk in immune cells, especially antigen-presenting cells. Syk inhibitors can take various forms, non-limiting examples of which include small molecules, nucleic acids, peptides, polypeptides, peptidomimetics etc.
In some embodiments, the Syk inhibitor is selected from compounds disclosed in U.S. Pat. No. 6,432,963, which is hereby incorporated by reference in its entirety. Exemplary compounds for example are encompassed by the definition set out between column 3, line 45 to column 6, line 22, and in particular a compound selected from the group consisting of 2-(2-aminoethylamino)-4-(3-methylanilino)pyrimidine-5-carboxamide, 2-(2-aminoethylamino)-4-(3-trifluoromethylanilino)pyrimidine-5-carboxamid-e, 2-(4-aminobutylamino)-4-(3-trifluoromethylanilino)pyrimidine-5-carboxam-ide, 2-(2-aminoethylamino)-4-(3-bromoanilino)pyrimidine-5-carboxamide, 2-(2-aminoethylamino)-4-(3-nitroanilino)pyrimidine-5-carboxamide, 2-(2-aminoethylamino)-4-(3,5-dimethylanilino)pyrimidine-5-carboxamide, 2-(2-aminoethylamino)-4-(2-naphthylamino)pyrimidine-5-carboxamide, 2-(cis-2-aminocyclohexylamino)-4-(3-methylanilino)pyrimidine-5-carboxamid-e, 2-(cis-2-aminocyclohexylamino)-4-(3-bromo-anilino)pyrimidine-5-carboxam-ide, 2-(cis-2-aminocyclohexylamino)-4-(3,5-dichloroanilino)pyrimidine-5-carboxamide and 2-(cis-2-aminocyclohexylamino)-4-(3,4,5-trimethoxyanilino)pyrimidine-5-carboxamide or a salt thereof. Methods for the synthesis of such compounds are set forth between column 6, line 43 to column 13, line 17.
Syk inhibitors, as employed in the present invention, also include compounds disclosed in U.S. Patent Application Publication No. US2004/0029902, which is hereby incorporated by reference in its entirety; including compounds encompassed by the definition set out between paragraphs [0109] and [0218], and in particular a compound selected from the group consisting of N2,N4-[(2,2-Dimethyl-4H-benzo[1,4]oxazin-3-one)-6-yl]-5-fluoro-2,4-pyrimi-dinediamine, N4-(3,4-Dichlorophenyl)-5-fluoro-N2-(indazoline-6-yl)-2,4-pyrimidinediami-ne, N4-(3,4-Ethylenedioxyphenyl)-5-fluoro-N2-(1-methyl-indazoline-5-yl)-2,4-p-yrimidinediamine, N2,N4-Bis(3-hydroxyphenyl)-5-fluoro-2,4-pyrimidinediamine, N2,N4-Bis(3,4-ethylenedioxyphenyl)-5-fluoro-2,4-pyrimidinediamine, N4-(1,4-Benzoxazin-6-yl)-5-fluoro-N2-[3-(N-methylamino)carbonylme-thyleneoxyphenyl]-2,4-pyrimidinediamine, N2,N4-Bis(3-aminophenyl)-5-fluoro-2,4-pyrimidinediamine, N4-(3,4-Ethylenedioxyphenyl)-5-fluoro-N2-[3-(N-methylamino)-carbonylmethy-leneoxyphenyl]-2,4-pyrimidinediamine, 5-Fluoro-N4-(3-hydroxyphenyl)-N2-[3-(N-methylamino)carbonylmethyleneoxyph-enyl]-2,4-pyrimidinediamine, N4-(3-Hydroxyphenyl)-5-trifluoromethyl-N2-[3-(N-methylamino)carbonylmethy-leneoxyphenyl]-2,4-pyrimidinediamine, 5-Fluoro-N4-[(1H)-indol-6-yl]-N2-[3-(N-methylamino)carbonylmethyleneoxyph-enyl]-2,4-pyrimidinediamine, 5-Fluoro-N4-(3-hydroxyphenyl)-N2-[3-(N-methylamino)carbonylmethyleneoxyph-enyl]-2,4-pyrimidinediamine, 5-Fluoro-N2-(3-methylaminocarbonylmethyleneoxyphenyl)-N4-[2-H-pyrido[3,2-b]-1,4-oxazin-3(4H)-one-6-yl]-2,4-pyrimidinediamine, N4-(3,4-Ethylenedioxyphenyl)-5-fluoro-N2-[3-(2-hydroxyethyl-amino)carbony-lmethyleneoxyphenyl]-2,4-pyrimidinediamine, 5-Fluoro-N4-(3-hydroxyphenyl)-N2-[3-(N-methylamino)carbonylmethyleneoxyph-enyl]-2,4-pyrimidinediamine, N2,N4-Bis(indol-6-yl)-5-fluoro-2,4-pyrimidinediamine, 5-Fluoro-N2-[2-(2-hydroxy-1,1-dimethylethylamino)carbonylbenzofuran-5-yl]--N4-(3-hydroxyphenyl)-2,4-pyrimidinediamine, N2-[3-(N2,3-Dihydroxypropylamino)carbonylmethyleneoxyphenyl]-N4-(3,4-ethy-lenedioxyphenyl)-5-fluoro-2,4-pyrimidinediamine, N2-(3,5-Dimethoxyphenyl)-N4-(3,4-ethylenedioxyphenyl)-5-fluoro-2,4-pyrimi-dinediamine, N4-(3,4-Ethylenedioxyphenyl)-5-fluoro-N2-[3-(1,3-oxazol-5-yl)phenyl]-2,4-pyrimidinediamine, N4-(3,4-Ethylenedioxyphenyl)-5-fluoro-N2-[3-(N-methylamino)-carbonylmethy-leneoxyphenyl]-2,4-pyrimidinediamine, 5-Fluoro-N2-(3-hydroxyphenyl)-N4-[4-(3-phenyl-1,2-4-oxadiazol-5-yl)methyl-eneoxyphenyl]-2,4-pyrimidinediamine, N4-(3,4-Ethylenedioxyphenyl)-5-fluoro-N2-(indazolin-6-yl)-2,4-pyrimidined-iamine, 5-Fluoro-N4-(3-hydroxyphenyl)-N2-(indazolin-6-yl)-2,4-pyrimidinediamine, N4-(3,4-Ethylenedioxyphenyl)-5-fluoro-N2-(1-methyl-indazoline-5-y-l)-2,4-pyrimidinediamine, 5-Fluoro-N4-(3-hydroxyphenyl)-N2-(1-methy-indazoline-5-yl)-2,4-pyrimidine-diamine, N4-(3,4-Ethylenedioxyphenyl)-5-fluoro-N2-[4-(3-phenyl-1,2,4-oxadiazol-5-y-l)methyleneoxyphenyl]-2,4-pyrimidinediamine, N4-(3,5-Dimethyl-4-hydroxyphenyl)-5-fluoro-N2-[3-[2-(N-morpholino)ethylen-eoxy]phenyl]-2,4-pyrimidinediamine, N4-(3,5-Dimethyl-4-hydroxyphenyl)-5-fluoro-N2-[3-[2-(N-morpholino)ethylox-y]phenyl]-2,4-pyrimidinediamine, N4-(3-Chloro-4-hydroxy-5-methylphenyl)-5-fluoro-N2-[3-[2-(N-morpholino)et-hyloxy]phenyl]-2,4-pyrimidinediamine, N2-(3-tert-Butylcarbonylaminophenyl)-N4-(3-hydroxyphenyl)-5-fluoro-2,4-py-rimidinediamine, N4-(3-tert-Butylphenyl)-N2-[3-(N-methylamino)carbonylmethyleneoxyphenyl]-5-fluoro-2,4-pyrimidinediamine, N4-(3-tert-Butylphenyl)-N2-[3-(N2,3-dihydroxypropylamino)carbonylmethylen-eoxyphenyl]-5-fluoro-2,4-pyrimidinediamine, N2-[3-(N2,3-Dihydroxypropylamino)carbonylmethyleneoxyphenyl]-5-fluoro-N4-(3-isopropylphenyl)-2,4-pyrimidinediamine, N4-[4-(Cyanomethyleneoxy)phenyl]-5-fluoro-N2-(3-hydroxyphenyl)-2,4-pyrimi-dinediamine, N4-(3,5-Dimethyl-4-hydroxyphenyl)-5-fluoro-N2-[3-(N-piperazino)carbonylme-thyleneoxyphenyl]-2,4-pyrimidinediamine, N4-(3,5-Dimethyl-4-hydroxyphenyl)-5-fluoro-N2-[3-[2-(N-piperazino)ethoxy]-phenyl]-2,4-pyrimidine-diamine bis hydrogenchloride salt, N4-(3,4-Ethylenedioxyphenyl)-5-fluoro-N2-[4-(2-hydroxyethyloxyl)phenyl]-2,-4-pyrimidinediamine, N4-(1,4-Benzoxazine-3-on-6-yl)-5-fluoro-N2-(3-hydroxyphenyl)-2,4-pyrimidi-nediamine, (+/−)-5-Fluoro-N2-[(N-methylacetamido-2)-3-phenoxy]-N4-(2-methyl-1,4-benz-oxazin-6-yl)-2,4-pyrimidinediamine, N2-(1,4-Benzoxazin-3-on-6-yl)-5-fluoro-N4-(3-hydroxyphenyl)-2,4-pyrimidin-ediamine, N4-(3-Chloro-4-trifluoromethoxyphenyl)-5-fluoro-N2-[3-(N-methylamino)carb-onylmethyleneoxyphenyl]-2,4-pyrimidinediamine, 5-Fluoro-N4-(3-hydroxy-4-methylphenyl)-N2-[3-[(N-methylamino)carbonylmeth-yleneoxy]phenyl]-2,4-pyrimidinediamine, 5-Fluoro-N4-(3-hydroxyphenyl)-N2-[4-methyl-3-[(N-methylamino)carbonylmeth-yleneoxy]phenyl]-2,4-pyrimidinediamine, 5-Fluoro-N4-(3-hydroxy-4-methoxyphenyl)-N2-[3-(N-methylamino)carbonylmeth-yleneoxyphenyl]-2,4-pyrimidinediamine, N4-(3-Chloro-4-methylphenyl)-5-fluoro-N2-[3-(N-methylamino)-carbonylmethy-leneoxyphenyl]-2,4-pyrimidinediamine, N4-(3-Chloro-4-methoxyphenyl)-5-fluoro-N2-[3-[(N-methylamino)carbonylmeth-yleneoxy]phenyl]-2,4-pyrimidinediamine, 5-Fluoro-N4-1(1H)-indol-5-yl]-N2-[3-[(N-methylamino)carbonylmethyleneoxy]-phenyl]-2,4-pyrimidinediamine, 5-Fluoro-N4-(3-hydroxyphenyl)-N2-[1-(methoxycarbonyl)methyl-indazoline-5-yl]-2,4-pyrimidinediamine, 5-Fluoro-N4-(3-hydroxyphenyl)-N2-[1-(3-hydroxypropyl)indazoline-6-yl]-2,4--pyrimidinediamine, N4-(3,4-Ethylenedioxyphenyl)-5-fluoro-N2-[1-(3-hydroxypropyl)indazoline-5--yl]-2,4-pyrimidinediamine, 5-Fluoro-N4-(3-hydroxyphenyl)-N2-[1-(3-hydroxypropyl)indazoline-5-yl]-2,4--pyrimidinediamine, 5-Fluoro-N2-[1-(3-hydroxypropyl)indazoline-5-yl]-N4-(4-isopropoxyphenyl)-2,4-pyrimidinediamine, N4-(3,4-Ethylenedioxyphenyl)-5-fluoro-N2-[1-[2(N-methylaminocarbonyl)ethy-l]-indazoline-5-yl]-2,4-pyrimidinediamine, 5-Fluoro-N4-(4-isopropoxyphenyl)-N2-[1-[2(N-methylaminocarbonyl)ethyl]-in-dazoline-5-yl]-2,4-pyrimidinediamine, N4-[(2,2-dimethyl-4H-benzo[1,4]oxazin-3-one)-6-yl]-5-fluoro-N2-[3-(methyl-aminocarbonylmethylene-oxy)phenyl]-2,4-pyrimidinediamine, N4-[(2,2-Dimethyl-4H-benzo[1,4]oxazin-3-one)-6-yl]-5-fluoro-N2-(1-methyli-ndazolin-5-yl)-2,4-pyrimidinediamine, N4-[(2,2-Difluoro-4H-benzo[1,4]oxazin-3-one)-6-yl]-5-fluoro-N2-[3-(methyl-aminocarbonylmethyleneoxy)phenyl]-2,4-pyrimidinediamine, N4-1 (2,2-Dimethyl-4H-5-pyridol-1,4]oxazin-3-one)-6-yl]-5-fluoro-N2-[3-(methyl-aminocarbonyl-methyleneoxy)phenyl]-2,4-pyrimidinediamine, 5-Fluoro-N2-(3-methylaminocarbonylmethyleneoxyphenyl)-N4-[2H-pyrido[3,2-b-]-1,4-oxazin-3(4H)-one-6-yl]-2,4-pyrimidinediamine, N4-(4-Amino-3,4-dihydro-2H-1-benzopyran-6-yl)-5-fluoro-N2-[3-(N-methylami-no)carbonylmethyleneoxyphenyl]-2,4-pyrimidinediamine, N4-(3-Chloro-4-hydroxy-5-methylphenyl)-5-fluoro-N2-[3-[2-(N-piperazino)et-hoxy]phenyl]-2,4-pyrimidinediamine, and N4-(3-Methylcarbonyloximephenyl)-5-fluoro-N2-[3-(N-methylamino)carbonylme-thyleneoxyphenyl]-2,4-pyrimidinediamine or a salt thereof. Such compounds can be synthesized for example in by methods set out between paragraphs [0218] and [0260] of U.S. Pat. Appl. Pub. No. 2004/0029902, which is hereby incorporated by reference in its entirety.
In other embodiments, Syk inhibitors are sepected from compounds described in U.S. Pat. Appl. Pub. No. 2010/0316649, which is hereby incorporated by reference in its entirety. Representative compounds of this type are represented by the following formulae Iz and Ib.
Compounds according to formula Iz are as set out below:
wherein
R1 is selected from aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heterocyclyl, substituted heterocyclyl, aralkyl, heteroaralkyl, hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, acyl, acylamino, and acyloxy;
R2a and R2b are independently selected from hydrogen, alkyl, substituted alkyl, acyl, acylamino, acyloxy, —SO-alkyl, —SO-aryl, —SO-heteroaryl, —SO2-alkyl, —SO2-aryl, —SO2-heteroaryl, aryl, substituted aryl, heteroaryl, heterocyclyl, aralkyl, and heteroaralkyl; and wherein either R2a or R2b is present;
R3 is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, halo, nitro, cyano, hydroxy, alkoxy, carboxyl, acyl, acylamino, aminoacyl, acyloxy, oxyacyl, amino, substituted amino, aryl, substituted aryl, heteroaryl, and substituted heteroaryl;
R5 is selected from hydrogen, alkyl, and substituted alkyl; and
R6 is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, acyl, acylamino, acyloxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aralkyl, heteroaralkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl, and substituted heterocyclyl;
or a salt or stereoisomer thereof.
Compounds according to formula Ib are shown below:
wherein
R1 is selected from aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heterocyclyl, substituted heterocyclyl, aralkyl, heteroaralkyl, hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, acyl, acylamino, and acyloxy;
R2a and R2b are independently selected from hydrogen, alkyl, substituted alkyl, acyl, acylamino, acyloxy, —SO-alkyl, —SO-aryl, —SO-heteroaryl, —SO2-alkyl, —SO2-aryl, —SO2-heteroaryl, aryl, substituted aryl, heteroaryl, heterocyclyl, aralkyl, and heteroaralkyl, and wherein either R2a or R2b is present;
R3 is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, halo, nitro, cyano, hydroxy, alkoxy, carboxyl, acyl, acylamino, aminoacyl, acyloxy, oxyacyl, amino, substituted amino, aryl, substituted aryl, heteroaryl, and substituted heteroaryl;
R4 is selected from hydrogen, alkyl, substituted alkyl, amino, or —NR5R6;
R5 is selected from hydrogen, alkyl, and substituted alkyl; and
R6 is selected from aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heterocyclyl, substituted heterocyclyl, aralkyl, heteroaralkyl, hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, acyl, acylamino, and acyloxy;
or a salt or stereoisomer thereof.
Illustrative examples of compounds according to formula Iz may be selected from: 3-(3,4-Dimethoxyphenylamino)-2-(2,4-dihydro-2-oxo-1H-benzo[d][1,3]oxazin-7-yl)-1H-imidazo[1,2-b]pyrazole-7-carbonitrile; N-(4-(3-(3,4-Dimethoxyphenylamino)-7-cyano-1H-imidazo[1,2-b]pyrazol-2-yl)-phenyl)acetamide; N-(4-(3-(3-(Trifluoromethyl)phenylamino)-7-cyano-1H-imidazo[1,2-b]pyrazol--2-yl)phenyl)acetamide; 3-(3,4-Dimethoxyphenylamino)-2-(5-methoxy-1H-indol-3-yl)-1H-imidazo[1,2-b-]pyrazole-7-carbonitrile; 3-(3,4-Dimethoxyphenylamino)-2-(1H-indol-5-yl)-1H-imidazo[1,2-b]pyrazole-7-carbonitrile; 3-(3,4-Dimethoxyphenylamino)-2-(1H-indol-6-yl)-1H-imidazo[1,2-b]pyrazole-7-carbonitrile; 3-(3,4-Dichlorophenylamino)-2-(2,4-dihydro-2-oxo-1H-benzo[d][1,3]oxazin-7--yl)-1H-imidazo[1,2-b]pyrazole-7-carbonitrile; 3-(3,4-Dimethoxyphenylamino)-2-(4-phenoxyphenyl)-1H-imidazo[1,2-b]pyrazol-e-7-carbonitrile; 3-(3-Cyanophenylamino)-2-(3,4-dihydro-3-oxo-2H-benzo[b][1,4]oxazin-6-yl)-1H-imidazo[1,2-b]pyrazole-7-carbonitrile; Methyl 3-(7-cyano-2-(3,4-dihydro-3-oxo-2H-benzo[b][1,4]oxazin-6-yl)-1H-imidazo[1-,2-b]pyrazol-3-ylamino)benzoate; 3-(2,4,4-Trimethylpentan-2-ylamino)-2-(3,4,5-trimethoxyphenyl)-1H-imidazo-[1,2-b]pyrazole-7-carbonitrile; N-(7-Cyano-2-(3,4,5-trimethoxyphenyl)-1H-imidazo[1,2-b]pyrazol-3-yl)-2,2,-2-trifluoroacetamide; Methyl 4-(7-cyano-2-(2,4-dihydro-2-oxo-1H-benzo[d][1,3]oxazin-7-yl)-1H-imidazo[1-,2-b]pyrazol-3-ylamino)benzoate; 3-(3-Methoxyphenylamino)-2-(3,4,5-trimethoxyphenyl)-1H-imidazo[1,2-b]pyra-zole-7-carbonitrile; 3-(3-Methoxyphenylamino)-2-(2,4-dihydro-2-oxo-1H-benzo[d][1,3]oxazin-7-yl-)-1H-imidazo[1,2-b]pyrazole-7-carbonitrile; 3-(3,4-Dimethoxyphenylamino)-2-(2,4-dihydro-2-oxo-1H-benzo[d][1,3]oxazin-7-yl)-6-methyl-1H-imidazo[1,2-b]pyrazole-7-carbonitrile; 3-Amino-2-(3,4,5-trimethoxyphenyl)-1H-imidazo[1,2-b]pyrazole-7-carbonitri-le; N-(7-Cyano-2-(3,4,5-trimethoxyphenyl)-1H-imidazo[1,2-b]pyrazol-3-yl)-3--fluoro-4-(trifluoromethyl)benzamide; N-(7-Cyano-2-(3,4,5-trimethoxyphenyl)-1H-imidazo[1,2-b]pyrazol-3-yl)benza-mide; N-(7-Cyano-2-(3,4,5-trimethoxyphenyl)-1H-imidazo[1,2-b]pyrazol-3-yl)--4-fluorobenz amide; N-(7-Cyano-2-(3,4,5-trimethoxyphenyl)-1H-imidazo[1,2-b]pyrazol-3-yl)-3-me-thoxybenzamide; Dichlorophenylamino)-2-(3,4,5-trimethoxyphenyl)-1H-imidazo[1,2-b]p-yrazole-7-carbonitrile; 3-(4-Bromophenylamino)-2-(3,4,5-trimethoxyphenyl)-1H-imidazo[1,2-b]pyrazo-le-7-carbonitrile; 3-(3,4-Dimethoxyphenylamino)-2-(6-methoxy-1H-indol-3-yl)-1H-imidazo[1,2-b-]pyrazole-7-carbonitrile; 3-(4-Morpholinophenylamino)-2-(3,4,5-trimethoxyphenyl)-1H-imidazo[1,2-b]p-yrazole-7-carbonitrile; 3-(3,4,5-Trimethoxyphenylamino)-2-(1H-indol-6-yl)-1H-imidazo[1,2-b]pyrazo-le-7-carbonitrile; 3-(4-Morpholinophenylamino)-2-(1H-indol-6-yl)-1H-imidazo[1,2-b]pyrazole-7--carbonitrile; 3-(3,4-Dimethoxyphenylamino)-2-(3,4,5-trimethoxyphenyl)-1H-imidazo[1,2-b]-pyrazole-7-carbonitrile; 3-(3,4,5-Trimethoxyphenylamino)-2-(3,4,5-trimethoxyphenyl)-1H-imidazo[1,2--b]pyrazole-7-carbonitrile; N-(3-(7-Cyano-2-(3,4,5-trimethoxyphenyl)-1H-imidazo[1,2-b]pyrazol-3-ylami-no)phenyl)acetamide; N-(3-(7-Cyano-2-(3,4-dihydro-3-oxo-2H-benzo[b][1,4]oxazin-6-yl)-1H-imidaz-o[1,2-b]pyrazol-3-ylamino)phenyl)acetamide; N-(3-(7-Cyano-2-(3,4-dimethoxyphenyl)-1H-imidazo[1,2-b]pyrazol-3-ylamino)-phenyl)acetamide; N-(3-(7-Cyano-2-(4-(methylthio)phenyl)-1H-imidazo[1,2-b]pyrazol-3-ylamino-)phenyl)acetamide; N-(4-(7-Cyano-3-(3-acetamidophenylamino)-1H-imidazo[1,2-b]pyrazol-2-yl)ph-enyl)acetamide; 3-(3-Methoxybenzylamino)-2-(3,4-dihydro-3-oxo-2H-benzo[b][1,4]oxazin-6-yl-)-1H-imidazo[1,2-b]pyrazole-7-carbonitrile; 3-(3,4-Dimethoxyphenylamino)-2-(2,4-difluorophenyl)-1H-imidazo[1,2-b]pyra-zole-7-carbonitrile; 3-(3,4-Dimethoxyphenylamino)-2-(3,4-difluorophenyl)-1H-imidazo[1,2-b]pyra-zole-7-carbonitrile; 3-(3,4-Dimethoxyphenylamino)-2-(2,3-dihydrobenzo[b][1,4]dioxin-7-yl)-1H-i-midazo[1,2-b]pyrazole-7-carbonitrile; N-(7-Cyano-2-(3,4,5-trimethoxyphenyl)-1H-imidazo[1,2-b]pyrazol-3-yl)-5-ph-enyl-1,3,4-oxadiazole-2-carboxamide; 3-(3,4-Dimethoxyphenethylamino)-2-(3,4-dihydro-3-oxo-2H-benzo[b][1,4]oxaz-in-6-yl)-1H-imidazo[1,2-b]pyrazole-7-carbonitrile; 3-(3,4-Dimethoxyphenethylamino)-2-(3,4,5-trimethoxyphenyl)-1H-imidazo[1,2--b]pyrazole-7-carbonitrile; N-(7-Cyano-2-(3,4,5-trimethoxyphenyl)-1H-imidazo[1,2-b]pyrazol-3-yl)-2-(3-,4-dimethoxyphenyl)acetamide; 3-(4-Morpholinophenylamino)-2-(3,4-dihydro-3-oxo-2H-benzo[b][1,4]oxazin-6--yl)-1H-imidazo[1,2-b]pyrazole-7-carbonitrile; 3-(3-Methoxyphenylamino)-2-(1H-indol-6-yl)-1H-imidazo[1,2-b]pyrazole-7-ca-rbonitrile; 3-(4-Bromophenylamino)-2-(3,4-dihydro-3-oxo-2H-benzo[b][1,4]oxazin-6-yl)-1H-imidazo[1,2-b]pyrazole-7-carbonitrile; 3-(3-Methoxybenzylamino)-2-(3,4,5-trimethoxyphenyl)-1H-imidazo[1,2-b]pyra-zole-7-carbonitrile; N-(3-(7-Cyano-2-(1H-indol-6-yl)-1H-imidazo[1,2-b]pyrazol-3-ylamino)phenyl-)acetamide; 3-(3,4-Dimethoxyphenylamino)-2-(4-(methylsulfonyl)phenyl)-1H-imidazo[1,2-b]pyrazole-7-carbonitrile; 3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-ylamino)-2-(3,4,5-trimethoxyphenyl)-1-H-imidazo[1,2-b]pyrazole-7-carbonitrile; 3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-ylamino)-2-(3,4-dihydro-3-oxo-2H-benz-o[b][1,4]oxazin-6-yl)-1H-imidazo[1,2-b]pyrazole-7-carbonitrile; 3-(3,4-Dimethoxyphenylamino)-2-(benzo[d][1,3]dioxol-6-yl)-1H-imidazo[1,2-b]pyrazole-7-carbonitrile; 3-(3,4-Dimethoxyphenylamino)-2-(3-fluoro-4-methoxyphenyl)-1H-imidazo[1,2-b]pyrazole-7-carbonitrile; 2-Bromo-N-(7-cyano-2-(3,4,5-trimethoxyphenyl)-1H-imidazo[1,2-b]pyrazol-3-yl)acetamide; N-(7-Cyano-2-(3,4,5-trimethoxyphenyl)-1H-imidazo[1,2-b]pyrazol-3-yl)-2-ph-enoxyacetamide; 3-(3,4-Dimethoxyphenylamino)-2-benzyl-1H-imidazo[1,2-b]pyrazole-7-carboni-trile; 2-(3,4-Dichlorophenyl)-N-(7-cyano-2-(3,4,5-trimethoxyphenyl)-1H-imi-dazo[1,2-b]pyrazol-3-yl)acetamide; 3-(3,4-Dimethoxyphenylamino)-2-(3,4,5-trifluorophenyl)-1H-imidazo[1,2-b]p-yrazole-7-carbonitrile; 3-(3,4-Dimethoxyphenylamino)-2-(3-chloro-4,5-dimethoxyphenyl)-1H-imidazo[-1,2-b]pyrazole-7-carbonitrile; 3-(3,4-Dimethoxyphenylamino)-2-(4-fluoro-3-methoxyphenyl)-1H-imidazo[1,2-b]pyrazole-7-carbonitrile; 3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-ylamino)-2-(1H-indol-6-yl)-1H-imidazo-[1,2-b]pyrazole-7-carbonitrile; N-(4-(7-Cyano-2-(3,4,5-trimethoxyphenyl)-1H-imidazo[1,2-b]pyrazol-3-ylami-no)phenyl)acetamide; N-(4-(7-Cyano-2-(6-methoxy-1H-indol-3-yl)-1H-imidazo[1,2-b]pyrazol-3-ylam-ino)phenyl)acetamide; N-(4-(7-Cyano-2-(2,3-dihydrobenzo[b][1,4]dioxin-7-yl)-1H-imidazo[1,2-b]py-razol-3-ylamino)phenyl)acetamide; 3-(3,4-Difluorophenylamino)-2-(3,4,5-trimethoxyphenyl)-1H-imidazo[1,2-b]p-yrazole-7-carbonitrile; tert-Butyl 4-(7-cyano-2-(3,4,5-trimethoxyphenyl)-1H-imidazo[1,2-b]pyrazol-3-ylamino)-benzylcarbamate; 3-(4-(Aminomethyl)phenylamino)-2-(3,4,5-trimethoxyphenyl)-1H-imidazo[1,2-b]pyrazole-7-carbonitrile; Methyl 3-(7-cyano-2-(3,4,5-trimethoxyphenyl)-1H-imidazo[1,2-b]pyrazol-3-ylamino)-benzoate; N-(4-(7-Cyano-2-(3,4-dihydro-3-oxo-2H-benzo[b][1,4]oxazin-6-yl)-1H-imidazo[1,2-b]pyrazol-3-ylamino)phenyl)acetamide; N-(4-(7-Cyano-2-(3,4-dimethoxyphenyl)-1H-imidazo[1,2-b]pyrazol-3-ylamino)-phenyl)acetamide; 3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-ylamino)-2-(3-hydroxy-4,5-dimethoxyph-enyl)-1H-imidazo[1,2-b]pyrazole-7-carbonitrile; Methyl 4-(7-cyano-2-(3-hydroxy-4,5-dimethoxyphenyl)-1H-imidazo[1,2-b]pyrazol-3-y-lamino)benzoate; N-(4-(7-Cyano-2-(3,4,5-trimethoxyphenyl)-1H-imidazo[1,2-b]pyrazol-3-ylami-no)benzyl)acetamide; tert-Butyl-4-((7-cyano-2-(3,4,5-trimethoxyphenyl)-1H-imidazo[1,2-b]pyrazo-l-3-ylamino)methyl)piperidine-1-carboxylate; 3-((Piperidin-4-yl)methylamino)-2-(3,4,5-trimethoxyphenyl)-1H-imidazo[1,2--b]pyrazole-7-carbonitrile; 3-(3-Fluoro-4-(4-(pyrrolidin-1-yl)piperidin-1-yl)phenylamino)-2-(3,4,5-tr-imethoxyphenyl)-1H-imidazo[1,2-b]pyrazole-7-carbonitrile; (S)-Methyl 2-(7-cyano-2-(3,4,5-trimethoxyphenyl)-1H-imidazo[1,2-b]pyrazol-3-ylamino)--3-phenylpropanoate; Methyl 3-(7-cyano-2-(3,4-dimethoxyphenyl)-1H-imidazo[1,2-b]pyrazol-3-ylamino)ben-zoate; Methyl 3-(2-(3-chloro-4,5-dimethoxyphenyl)-7-cyano-1H-imidazo[1,2-b]pyrazol-3-yl-amino)benzoate; 3-(3,4,5-Trimethoxyphenylamino)-2-(4-morpholinophenyl)-1H-imidazo[1,2-b]p-yrazole-7-carbonitrile; 3-(4-Bromophenylamino)-2-(4-morpholinophenyl)-1H-imidazo[1,2-b]pyrazole-7--carbonitrile; 4-(3-(3,4,5-Trimethoxyphenylamino)-7-cyano-1H-imidazo[1,2-b]pyrazol-2-yl)-benzamide; 3-Amino-2-(3,4,5-trimethoxyphenyl)-5-(4-methoxyphenyl)-5H-imida-zo[1,2-b]pyrazole-7-carbonitrile; N-(4-(7-Cyano-2-(3,4,5-trimethoxyphenyl)-5H-imidazo[1,2-b]pyrazol-3-ylami-no)benzyl)nicotinamide; Methyl 3-(2-(4-((methoxycarbonyl)methoxy)-3-methoxyphenyl)-7-cyano-1H-imidazo[1,-2-b]pyrazol-3-ylamino)benzoate; 3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-ylamino)-2-(4-((methoxycarbonyl)metho-xy)-3-methoxyphenyl)-1H-imidazo[1,2-b]pyrazole-7-carbonitrile; 2-(5-(7-Cyano-3-(3-(methoxycarbonyl)phenylamino)-1H-imidazo[1,2-b]pyrazol--2-yl)-2-methoxyphenoxy)acetic acid; 3-(4-Fluoro-3-methoxyphenylamino)-2-(3,4,5-trimethoxyphenyl)-1H-imidazo[1-,2-b]pyrazole-7-carbonitrile; 6-(4-Chlorophenyl)-2-(3,4-dimethoxyphenyl)-5H-imidazo[1,2-b]pyrazole-7-ca-rbonitrile; N-(4-(7-Cyano-2-(3,4,5-trimethoxyphenyl)-5H-imidazo[1,2-b]pyrazol-3-ylami-no)benzyl)-3,4-dimethoxybenzamide; N-(4-(7-Cyano-2-(3,4,5-trimethoxyphenyl)-5H-imidazo[1,2-b]pyrazol-3-ylami-no)benzyl)-3-(4-hydroxyphenyl)propanamide; N-(4-(7-Cyano-2-(3,4,5-trimethoxyphenyl)-5H-imidazo[1,2-b]pyrazol-3-ylami-no)benzyl)-3-(piperidin-1-yl)propanamide; N-(4-(7-Cyano-2-(3,4,5-trimethoxyphenyl)-5H-imidazo[1,2-b]pyrazol-3-ylami-no)benzyl)-4-cyanobenzamide; N-(4-(7-Cyano-2-(3,4,5-trimethoxyphenyl)-5H-imidazo[1,2-b]pyrazol-3-ylami-no)benzyl)-1-methylpiperidine-4-carboxamide; N-(4-(7-Cyano-2-(3,4,5-trimethoxyphenyl)-5H-imidazo[1,2-b]pyrazol-3-ylami-no)benzyl)-1H-indazole-3-carboxamide; N-(4-(7-Cyano-2-(3,4,5-trimethoxyphenyl)-5H-imidazo[1,2-b]pyrazol-3-ylami-no)benzyl)-1,6-dihydro-6-oxopyridine-3-carboxamide; 3-((1-Nicotinoylpiperidin-4-yl)methylamino)-2-(3,4,5-trimethoxyphenyl)-5H--imidazo[1,2-b]pyrazole-7-carbonitrile; 4-(3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-ylamino)-7-cyano-1H-imidazo[1,2-b]-pyrazol-2-yl)benzamide; 4-(3-(4-Bromophenylamino)-7-cyano-1H-imidazo[1,2-b]pyrazol-2-yl)benzamide-; Methyl 2-(4-(7-cyano-3-(3,4-dimethoxyphenylamino)-1H-imidazo[1,2-b]pyraz-ol-2-yl)-2-methoxyphenoxy)acetate; 3-(3,4-Dimethoxyphenylamino)-2-(3-hydroxy-4,5-dimethoxyphenyl)-1H-imidazo-[1,2-b]pyrazole-7-carbonitrile; 2-(4-(2-Hydroxyethoxy)-3-methoxyphenyl)-3-(3,4-dimethoxyphenylamino)-1H-i-midazo[1,2-b]pyrazole-7-carbonitrile; 3-(4-Fluoro-3-methoxyphenylamino)-2-(3,4-dimethoxyphenyl)-5H-imidazo[1,2-b]pyrazole-7-carbonitrile; 4-(3-(3,4-Dimethoxyphenylamino)-7-cyano-1H-imidazo[1,2-b]pyrazol-2-yl)ben-zamide; 3-(3,4-Dimethoxyphenylamino)-2-(4-morpholinophenyl)-1H-imidazo[1,2--b]pyrazole-7-carbonitrile; 3-(3,4-Dimethoxyphenylamino)-2-(3-morpholinophenyl)-1H-imidazo[1,2-b]pyra-zole-7-carbonitrile; 3-(3,4-Dimethoxyphenylamino)-2-(3-(cyclopentyloxy)-4-methoxyphenyl)-1H-im-idazo[1,2-b]pyrazole-7-carbonitrile; 3-(3,4-Dimethoxyphenylamino)-2-(4-(2-pyrrolidin-1-yl)ethoxy)-1H-imidazo[1-,2-b]pyrazole-7-carbonitrile; 2-(4-(2-Methoxyethoxy)-3-methoxyphenyl)-3-(3,4-dimethoxyphenylamino)-1H-i-midazo[1,2-b]pyrazole-7-carbonitrile; 3-(4-Fluoro-3-methoxyphenylamino)-2-(3,4-dihydro-3-oxo-2H-benzo[b][1,4]ox-azin-6-yl)-1H-imidazo[1,2-b]pyrazole-7-carbonitrile; 3-(3-(Trifluoromethoxy)phenylamino)-2-(3,4,5-trimethoxyphenyl)-1H-imidazo-[1,2-b]pyrazole-7-carbonitrile; 3-(3-Chloro-4-methoxyphenylamino)-2-(3,4,5-trimethoxyphenyl)-1H-imidazo[1-,2-b]pyrazole-7-carbonitrile; 3-(3,4-Dimethoxyphenylamino)-2-(3-hydroxyphenyl)-1H-imidazo[1,2-b]pyrazol-e-7-carbonitrile; Methyl 2-(4-(7-cyano-3-(3,4-dimethoxyphenylamino)-1H-imidazo[1,2-b]pyrazol-2-yl)-phenoxy)acetate; N-(3-(7-Cyano-3-(3,4-dimethoxyphenylamino)-1H-imidazo[1,2-b]pyrazol-2-yl)-phenyl)methanesulfonamide; 3-(3-(Cyclopentyloxy)-4-methoxyphenylamino)-2-(3,4,5-trimethoxyphenyl)-1H--imidazo[1,2-b]pyrazole-7-carbonitrile; 2-(4-(2-Hydroxyethoxy)-3-methoxyphenyl)-3-(4-fluoro-3-methoxyphenylamino)--1H-imidazo[1,2-b]pyrazole-7-carbonitrile; 4-(3-(4-Fluoro-3-methoxyphenylamino)-7-cyano-1H-imidazo[1,2-b]pyrazol-2-y-l)benzamide; 3-(4-Fluoro-3-methoxyphenylamino)-2-(3-hydroxy-4,5-dimethoxyphenyl)-1H-im-idazo[1,2-b]pyrazole-7-carbonitrile; 3-(4-Fluoro-3-methoxyphenylamino)-2-(3-(cyclopentyloxy)-4-methoxyphenyl)-1H-imidazo[1,2-b]pyrazole-7-carbonitrile; 3-(4-Fluoro-3-methylphenylamino)-2-(3,4,5-trimethoxyphenyl)-1H-imidazo[1,-2-b]pyrazole-7-carbonitrile; 3-(3-Fluoro-4-methylphenylamino)-2-(3,4,5-trimethoxyphenyl)-1H-imidazo[1,-2-b]pyrazole-7-carbonitrile; 2-(4-(2-Methoxyethoxy)-3-methoxyphenyl)-3-(4-fluoro-3-methoxyphenylamino)--1H-imidazo[1,2-b]pyrazole-7-carbonitrile; Methyl 2-(4-(7-cyano-3-(4-fluoro-3-methoxyphenylamino)-1H-imidazo[1,2-b]pyrazol-2-yl)-2-methoxyphenoxy)acetate; 2-(4-(2-Morpholinoethoxy)phenyl)-3-(3,4-dimethoxyphenylamino)-1H-imidazo[-1,2-b]pyrazole-7-carbonitrile; 2-(5-(7-Cyano-3-(3,4-dimethoxyphenylamino)-1H-imidazo[1,2-b]pyrazol-2-yl)--2-methoxyphenoxy)acetamide; 3-(3-Isopropoxy-4-methoxyphenylamino)-2-(3,4,5-trimethoxyphenyl)-1H-imida-zo[1,2-b]pyrazole-7-carbonitrile; 3-(3-Fluoro-4-methoxyphenylamino)-2-(3,4,5-trimethoxyphenyl)-1H-imidazo[1-,2-b]pyrazole-7-carbonitrile; 3-(4-(Cyclopentyloxy)-3-methoxyphenylamino)-2-(3,4,5-trimethoxyphenyl)-1H--imidazo[1,2-b]pyrazole-7-carbonitrile; 3-(4-(2-(Pyrrolidin-1-yl)ethoxy)-3-methoxyphenylamino)-2-(3,4,5-trimethoxyphenyl)-1H-imidazo[1,2-b]pyrazole-7-carbonitrile; 3-(4-Fluoro-3-(trifluoromethyl)phenylamino)-2-(3,4,5-trimethoxyphenyl)-1H--imidazo[1,2-b]pyrazole-7-carbonitrile; 3-(4-(Trifluoromethoxy)phenylamino)-2-(3,4,5-trimethoxyphenyl)-1H-imidazo-[1,2-b]pyrazole-7-carbonitrile; 3-(4-Chloro-3-methoxyphenylamino)-2-(3,4,5-trimethoxyphenyl)-1H-imidazo[1-,2-b]pyrazole-7-carbonitrile; 3-(4-Fluoro-3-isopropoxyphenylamino)-2-(3,4,5-trimethoxyphenyl)-1H-imidaz-o[1,2-b]pyrazole-7-carbonitrile; 3-(3-Fluoro-4-(pyrrolidin-1-yl)phenylamino)-2-(3,4,5-trimethoxyphenyl)-1H--imidazo[1,2-b]pyrazole-7-carbonitrile; 2-(4-(7-Cyano-3-(3,4-dimethoxyphenylamino)-1H-imidazo[1,2-b]pyrazol-2-yl)--2-methoxyphenoxy)acetamide; 3-(3,4-Dimethoxyphenylamino)-2-(4-methoxy-3,5-dimethylphenyl)-1H-imidazo[-1,2-b]pyrazole-7-carbonitrile; 3-(3,4-Dihydro-3-oxo-2H-benzo[b][1,4]oxazin-6-ylamino)-2-(3,4,5-trimethox-yphenyl)-1H-imidazo[1,2-b]pyrazole-7-carbonitrile; 2-(5-(7-Cyano-3-(4-fluoro-3-methoxyphenylamino)-1H-imidazo[1,2-b]pyrazol-2-yl)-2-methoxyphenoxy)acetamide; 2-(4-(7-Cyano-3-(4-fluoro-3-methoxyphenylamino)-1H-imidazo[1,2-b]pyrazol-2-yl)-2-methoxyphenoxy)acetamide; 2-(4-(7-Cyano-3-(3,4-dimethoxyphenylamino)-1H-imidazo[1,2-b]pyrazol-2-yl)--2-methoxyphenoxy)-N-cyclopropylacetamide; 3-(3-Chloro-4-isopropoxyphenylamino)-2-(3,4,5-trimethoxyphenyl)-1H-imidaz-o[1,2-b]pyrazole-7-carbonitrile; 3-(3,5-Dimethoxyphenylamino)-2-(3,4,5-trimethoxyphenyl)-1H-imidazo[1,2-b]-pyrazole-7-carbonitrile; 3-(3,5-Difluoro-4-methoxyphenylamino)-2-(3,4,5-trimethoxyphenyl)-1H-imida-zo[1,2-b]pyrazole-7-carbonitrile; 3-(3-Ethoxy-4-fluorophenylamino)-2-(3,4,5-trimethoxyphenyl)-1H-imidazo[1,-2-b]pyrazole-7-carbonitrile; 3-(3-(Cyclopentyloxy)-4-fluorophenylamino)-2-(3,4,5-trimethoxyphenyl)-1H-imidazo[1,2-b]pyrazole-7-carbonitrile; N-(3-(7-Cyano-2-(3,4,5-trimethoxyphenyl)-1H-imidazo[1,2-b]pyrazol-3-ylami-no)benzyl)nicotinamide; N-(3-(7-Cyano-2-(3,4,5-trimethoxyphenyl)-1H-imidazo[1,2-b]pyrazol-3-ylami-no)benzyl)picolinamide; N-(3-(7-Cyano-2-(3,4,5-trimethoxyphenyl)-1H-imidazo[1,2-b]pyrazol-3-ylami-no)benzyl)isonicotinamide; N-(4-(7-Cyano-2-(3,4,5-trimethoxyphenyl)-1H-imidazo[1,2-b]pyrazol-3-ylami-no)benzyl)picolinamide; N-(4-(7-Cyano-2-(3,4,5-trimethoxyphenyl)-1H-imidazo[1,2-b]pyrazol-3-ylami-no)benzyl)isonicotinamide; N-(4-(7-Cyano-2-(3,4,5-trimethoxyphenyl)-1H-imidazo[1,2-b]pyrazol-3-ylami-no)benzyl)-6-cyanopyridine-3-carboxamide; N-(4-(7-Cyano-2-(3,4,5-trimethoxyphenyl)-1H-imidazo[1,2-b]pyrazol-3-ylami-no)benzyl)-2-methylpyridine-3-carboxamide; N-(4-(7-Cyano-2-(3,4,5-trimethoxyphenyl)-1H-imidazo[1,2-b]pyrazol-3-ylami-no)benzyl)-2-methoxypyridine-3-carboxamide; N-(4-(7-Cyano-2-(3,4,5-trimethoxyphenyl)-1H-imidazo[1,2-b]pyrazol-3-ylami-no)benzyl)-6-methylpyridine-3-carboxamide; N-(4-(7-Cyano-2-(3,4,5-trimethoxyphenyl)-1H-imidazo[1,2-b]pyrazol-3-ylami-no)benzyl)-4-(trifluoromethyl)pyridine-3-carboxamide; N-(4-(7-Cyano-2-(3,4,5-trimethoxyphenyl)-1H-imidazo[1,2-b]pyrazol-3-ylami-no)benzyl)-6-(trifluoromethyl)pyridine-3-carboxamide; and 3-(3-Fluoro-4-(methylthio)phenylamino)-2-(3,4,5-trimethoxyphenyl)-1H-imid-azo[1,2-b]pyrazole-7-carbonitrile.
In still other embodiments, the Syk inhibitors may be selected from aminopyrimidine compounds as disclosed for example in U.S. Pat. Appl. Pub. No. 2011/0245205, which is hereby incorporated by reference in its entirety. Non-limiting inhibitor compounds of this type are represented by the formula (IV) or a pharmaceutically acceptable salt thereof:
wherein:
R1 is selected from the group consisting of (a) hydrogen, (b) halogen, (c) CN, (d) C1-6 alkyl optionally substituted with one or more groups independently selected from the group consisting of ORa, C3-6cycloalkyl, and halogen, (e) C2-6 alkenyl optionally substituted with OC1-6alkyl, (f) C2-6 alkynyl, (g) C3-6 cycloalkyl, (h) OH, (i) —O—C1-6 alkyl optionally substituted with one or more groups independently selected from (i) aryl, (ii) 5- or 6-membered heteroaryl optionally substituted with one or more groups independently selected from C1-6 alkyl, (iii) 4- to 8-membered heterocyclyl optionally substituted with one or more groups independently selected from oxo, halogen, C1-6 alkyl, (iv) —CO2Ra, (v) —CONRbRc, (vi) —NRbRc, and (vii) —ORa (j) -A-X, wherein A is a bond or O, X is selected from the group consisting of (i) 4- to 8-membered heterocyclyl optionally substituted with one or more groups independently selected from halogen, C1-6 alkyl, —C1-6 haloalkyl, —C1-6 hydroxyalkyl, CORa, CO2Ra, (ii) C3-6 cycloalkyl optionally substituted with one or more groups independently selected from C1-6 alkyl, —ORa, —CO2Ra, —NRbRc, (iii) heteroaryl optionally substituted with a benzyl which is optionally substituted with ORa, (k) O—CH2C.ident.C-pyrimidinyl, (l) —S(O)n—C1-6 alkyl, (m) —CORa, (n) —CO2Ra, (o) —CONRbRc, (p) —NRbRc;
R2 is selected from the group consisting of (a) H, (b) halogen, (c) C1-6 alkyl, (d) O—C1-6 alkyl, (e) C1-6 haloalkyl and (f) O—C1-6 haloalkyl; or R1 and R2 on adjacent carbon atoms together represent (CH2)3-4;
R3 is H, halogen, ORa, or C1-4allyl;
R4 is selected from the group consisting of (a) H, (b) halogen, (c) C1-6 alkyl optionally substituted with one or more groups independently selected from (i) halogen, (ii) ORa, (iii) OC(O)Ra, (iv) NRbRc, (v) NHC(O)Ra, and (vi) NHC(O)NHRb, (d) C2-6 alkenyl, (e) C2-6 alkynyl, (f) C3-6 cycloalkyl (g) ORa, (h) NO2, (i) NRbRc, (j) NHC(O)Ra, (k) NHC(O)NHRb, (l) NHC(O)NHC(O)NRbRc;
R5 is selected from the group consisting of (a) H, (b) halogen, (c) C1-8alkyl, C2-6alkenyl, C2-6 alkynyl, each of which is optionally substituted with one or more groups independently selected from Ry, (d) C3-12 carbocycle, or a carbon-linked 3- to 12-membered heterocyclyl each optionally substituted with one or more groups independently selected from Rz, (e) heteroaryl optionally substituted with C1-3 alkyl (optionally substituted with one or more OH or CN or heterocycle); (f) —C(O)Ra, (g) —C(O)2Ra, and (h) —C(O)NRbRc;
R5(i) is selected from the group consisting of H and C1-3 alkyl;
Ra is selected from the group consisting of (a) H, (b) C1-6 alkyl optionally substituted with one or more groups independently selected from (i) halogen, (ii) CN, (iii) OH, (iv) OC1-4alkyl, (v) heterocyclyl optionally substituted with oxo, (vi) C(O)C1-6alkyl optionally substituted with OH, (vii) CO2H, (viii) CO2C1-6alkyl, (ix) CONRb(i)Rc(i), (x) SO2C1-6alkyl, (xi) —NRb(i)Rc(i), (xii) NRb(i) C(O)NRb(i)Rc(i); (xiii) phenyl, and (xiv) heteroaryl optionally substituted with OH, (c) C2-6alkenyl, (d) C3-6 cycloalkyl optionally substituted with one or more groups independently selected from (i) OH, (ii) CO2H, (iii) CO2C1-6alkyl, (iv) CONRb(i)Rc(i) (e) phenyl optionally substituted with one or more groups independently selected from (i) C2-6alkynyl, (ii) CN, (iii) halogen, (iv) OH, (vi) OC(O)C1-6alkyl, (vii) CO2H, (viii) CO2C1-6alkyl, (f) heteroaryl optionally substituted with one or more groups independently selected from C1-6alkyl, C1-6haloalkyl, (CH2)1-8CO2H, OH, halogen, phenyl optionally substituted with CO2H, and (g) heterocyclyl optionally substituted with oxo;
Rb and Rc are independently selected from the group consisting of (a) H, (b) C1-6 alkyl optionally substituted with one or more groups independently selected from (i) ORa, (ii) halogen, (iii) heterocyclyl optionally substituted with oxo, OH, C1-6 alkyl (optionally substituted with OH), (iv) C3-6 cycloalkyl optionally substituted with one or two groups selected from C1-4alkyl, CH2OH, CONRb(i)Rc(i), and CO2Ra, (v) heteroaryl optionally substituted with C1-6alkyl optionally substituted with OH, CO2H or heteroaryl optionally substituted with a heteroaryl, (vi) SO2NRb(i)Rc(i), (vii) SO2C1-4alkyl, (viii) CONRb(i)Rc(i), (ix) NRb(i)Rc(i), (x) CO2Ra, (xi) aryl optionally substituted with one or more groups selected from halogen, ORa, C1-6alkyl (optionally substituted with halogen, heterocycle (optionally substituted with oxo), or ORa), SO2NH2, and heteroaryl optionally substituted with CH2OH (xii) SO3H, (xiii) NRb(i) CONRb(i)Rc(i), (xiv) CN, and (xv) NHC(O)Ra, (c) C3-6 alkenyl optionally substituted with F; (d) C3-6 cycloalkyl (optionally fused to a benzene ring) optionally substituted with one or more groups independently selected from (i) C1-4alkyl, (ii) ORa, (iii) CH2OH, (iv) CO2Ra, and (v) CONRb(i)Rc(i), (e) aryl optionally substituted with one or two groups independently selected from (i) C1-6alkyl (optionally substituted with ORa), (ii) CN, (iii) ORa, (iv) halogen, and (v) OCOC1-4alkyl; (f) heteroaryl optionally substituted with one or more groups independently selected from (i) ORa, (ii) CO2Ra and (iii) C1-6 alkyl optionally substituted with OH, (g) heterocyclyl optionally substituted with one or more groups independently selected from (i) oxo, (ii) OH and (iii) C1-6 alkyl; or
Rb, Rc and the nitrogen atom to which they are attached together form a 5-, 6- or 7-membered heterocycle having 0 or 1 additional heteroatom selected from 0, P(O)(C1-6alkyl), S(O)n and N—Rx, and optionally substituted with one or more groups independently selected from (a) oxo, (b) thioxo, (c) C1-6 alkyl optionally substituted with one or more groups independently selected from (i) ORa, (ii) CO2Ra, (iii) OP(O)(C1-6alkyl)2, (iv) aryl, and (v) halogen, (d) ORa, (e) C(O)Ra, (f) C(O)2Ra, (g) CONRb(i)Rc(i), (h) P(O)(OH)2, (i) SO2Ra, and (j) CN, or Rb, Rc and the nitrogen atom to which they are attached together form
wherein W is CH or N;
Rb(i) and Rc(i) are independently selected from the group consisting of (a) H and (b) C1-6 alkyl optionally substituted with OH, CO2H or CO2C1-6alkyl; or
Rb(i), Rc(i) and the nitrogen atom to which they are attached together form a 5- or 6-membered heterocycle having 0 or 1 additional heteroatom selected from O, S and N—Rx, and optionally substituted with one or more groups independently selected from oxo;
Ru is selected from the group consisting of (a) C1-6 alkyl optionally substituted with one to three groups selected from halogen, OH, SO2Ra, CONRbRc, NRbRc, phenyl, heterocyclyl and heteroaryl, (b) C3-8 cycloalkyl optionally substituted with OH, CO2Ra, —CONH2, (c) heterocycle optionally substituted with oxo, (d) aryl optionally substituted with C2-6alkynyl, CN, halogen, ORa, (e) heteroaryl optionally substituted with OH;
Rx is selected from the group consisting of (a) H, (b) C1-6 alkyl optionally substituted with heterocycle, (c) phenyl optionally substituted with OH or OC1-4alkyl, (d) —C(O)—C1-6 alkyl, (e) —C(O)2—C1-6 alkyl, (f) —C(O)NH2, —C(O)NH—C1-6 alkyl, —C(O)N(C1-6 alkyl)2, (g) —C(O)2NHC(O)NH2, —C(O)2NHC(O)NH—C1-6 alkyl, —C(O)2NHC(O)N(C1-6 alkyl)2, (h) —SO2—C1-6 alkyl (optionally substituted with halogen), —SO2-heteroaryl (optionally substituted with alkyl), (i) —S(O)2NH2, —S(O)2NH—C1-6 alkyl, —S(O)2N(C1-6 alkyl)2, (j) —SO2NHC(O)2—C1-6 alkyl;
Ry is selected from the group consisting of (a) aryl optionally substituted with one or more groups independently selected from (i) halogen, (ii) C1-6allyl optionally substituted with OH or CO2Ra, (iii) C2-6alkenyl optionally substituted with CO2Ra, (iv) phenyl optionally substituted with CO2Ra, (v) CORa, (vi) CO2Ra, (vii) CONRbRc, (viii) ORa, (ix) S(O)nRa, (x) SO2NRbRc, (xi) SO2NHC(O)Ra, (xii) NO2, and (xiii) NHC(O)Ra, (b) heteroaryl optionally substituted with one or more groups independently selected from (i) halogen, (ii) C1-6 alkyl optionally substituted with CO2Ra, (iii) C3-6 cycloalkyl, (iv) aryl optionally substituted with CO2Ra, (v) CONRbRc, (vi) ORa, (vii) SO2Ra, and (viii) CO2Ra, (c) C3-8 cycloalkyl optionally substituted with one or more groups independently selected from (i) C1-6 alkyl, (ii) CO2Ra, and (iii) NRbRc, (d) C6-8cycloalkenyl (optionally substituted with CO2Ra), (e) halogen, (f) CN, (g) —C(O)Ra, (h) —C(O)2Ra, (i) C(O)CO2Ra, (j) —C(O)NRbRc, (k) —C(O)NHC(O)NRbRc, (l) —ORa, (m) —OC(O)Ra, (n) —NRbRc, (o) —NHC(O)Ru, (p) —NHC(O)NRbRc, (q) —NHC(O)NHC(O)NH2, (r) —NHSO.sub.mRa (s) —NHSO2NRbRc, (t) SOnRa, (u) —SO2NRbRc, (v) —SO2NHC(O)Ra, (w) —SO2NHC(O)2Ra, (x) SO3H, (y) —P(O)(ORa)2, and (z) CONHOH; (aa) heterocyclyl optionally substituted with one or more groups independently selected from oxo, thioxo, C1-6alkyl, and CO2Ra;
Rz is selected from the group consisting of (a) a group selected from Ry, (b) C1-6 alkyl optionally substituted with one or more groups independently selected from halogen, NRbRc, ORa, CN, phenyl (optionally substituted with C1-6alkanoic acid), CONRbRc, and —CO2Ra, (c) oxo, and (d) NORa, m is 1 or 2, n is 0, 1 or 2.
Representative compounds according to formula IV include but are not limited to: (1R,4S)-4-[5-(3-cyclopropyl-5-{[4-(trifluoromethyl)pyrimidin-2-yl]amino}p-henyl)-1,3-thiazol-2-yl]-4-hydroxy-2,2-dimethylcyclohexanecarboxylic acid; (1S,4R)-4-[5-(3-cyclopropyl-5-{[4-(trifluoromethyl)pyrimidin-2-yl]amino}p-henyl)-1,3-thiazol-2-yl]-4-hydroxy-2,2-dimethylcyclohexanecarboxylic acid; (1S,4R)-4-hydroxy-2,2-dimethyl-4-{5-[3-methyl-5-(4-methyl-pyrimidin-2-yla-mino)-phenyl]-1,3-thiazol-2-yl}-cyclohexanecarboxylic acid; (1S,4R)-4-[5-(3-{[4-(difluoromethyl)pyrimidin-2-yl]amino}-5-methylphenyl)--1,3-thiazol-2-yl]-4-hydroxy-2,2-dimethylcyclohexanecarboxylic acid; trans-4-hydroxy-4-[5-(3-methyl-5-{[4-(trifluoromethyl)pyrimidin-2-yl]amin-o}phenyl)-1,3-thiazol-2-yl]cyclohexanecarboxylic acid; cis-4-hydroxy-4-[5-(3-methyl-5-{[4-(trifluoromethyl)pyrimidin-2-yl]amino}-phenyl)-1,3-thiazol-2-yl]cyclohexanecarboxylic acid; 5-hydroxy-5-[5-(3-methyl-5-{[4-(trifluoromethyl)pyrimidin-2-yl]amino}phen-yl)-1,3-thiazol-2-yl]azepan-2-one; cis-4-[(hydroxyacetyl)amino]-1-[5-(3-methyl-5-{[4-(trifluoromethyl)pyrimi-din-2-yl]amino}phenyl)-1,3-thiazol-2-yl]cyclohexanecarboxamide; and (1S,4R)-4-hydroxy-2,2-dimethyl-4-[5-(3-methyl-5-{[4-(trifluoromethyl) pyri-midin-2-yl]amino}phenyl)-1,3-thiazol-2-yl]-N-[3-(2-oxopyrrolidin-1-yl)propyl]cyclohe-xanecarboxamide; (1S,4R)-4-hydroxy-2,2-dimethyl-4-[5-(3-methyl-5-{[4-(trifluoromethyl)pyrimidin-2-yl]amino}-phenyl)-1,3-thiazol-2-yl]cy-clohexanecarboxylic acid; (1R,48)-4-hydroxy-2,2-dimethyl-4-[5-(3-methyl-5-{[4-(trifluoromethyl)pyri-midin-2-yl]amino}-phenyl)-1,3-thiazol-2-yl]cyclohexanecarboxylic acid; (1S,4S)-4-hydroxy-2,2-dimethyl-4-[5-(3-methyl-5-{[4-(trifluoromethyl)pyri-midin-2-yl]amino}-phenyl)-1,3-thiazol-2-yl]cyclohexanecarboxylic acid; (1R,4R)-4-hydroxy-2,2-dimethyl-4-[5-(3-methyl-5-{[4-(trifluoromethyl)pyri-midin-2-yl]amino}-phenyl)-1,3-thiazol-2-yl]cyclohexanecarboxylic acid; (1RAS)-4-hydroxy-2,2-dimethyl-4-[5-(3-methyl-5-{[4-(trifluoromethyl)pyrim-idin-2-yl]amino}phenyl)-1,3-thiazol-2-yl]cyclohexanecarboxamide; (1S,4R)-4-hydroxy-2,2-dimethyl-4-[5-(3-methyl-5-{[4-(trifluoromethyl)pyri-midin-2-yl]amino}phenyl)-1,3-thiazol-2-yl]cyclohexanecarboxamide; (1S,4R) 4-{5-[3-({4-[(1R)-1-fluoroethyl]pyrimidin-2-yl}amino)-5-methylphenyl]-1,3--thiazol-2-yl}-4-hydroxy-2,2-dimethylcyclohexanecarboxylic acid; (1S,4R) 4-{5-[3-({4-[(1S)-1-fluoroethyl]pyrimidin-2-yl}amino)-5-methylphenyl]-1,3--thiazol-2-yl}-4-hydroxy-2,2-dimethylcyclohexanecarboxylic acid; or a pharmaceutically acceptable salt thereof.
3.2.6 Immunomodulating Particle EmbodimentsIn some embodiments, at least one aggrecan antigen as described for example in Section 3.2.1 together with one or more inhibitors as described above (e.g., at least one NF-κB inhibitor as described for example in Section 3.2.3, and/or at least one mTOR inhibitor as described for example in Section 3.2.4, and/or at least one Syk inhibitor as described for example in Section 3.2.5), or alternatively at least one or aggrecan APL as described for example in Section 3.2.2 (collectively referred to herein as “the bioactive agents”) are provided in particulate form, which facilitate in vivo or in vitro delivery of the antigen and inhibitor to antigen-presenting cells. The inhibitor(s) and the antigen(s) or the APLs may be contained in or otherwise associated with the same particle or different particles. A variety of particles may be used in the practice of the present invention, including but not limited to, liposomes, micelles, lipidic particles, ceramic/inorganic particles and are typically selected from nanoparticles and microparticles. The particles are suitably sized for being taken up (e.g., by phagocytosis or endocytosis) by antigen-presenting cells. In illustrative examples, the particles have a dimension of less than about 100 μm, more suitably in the range of less than or equal to about 500 nm, although the particles may be as large as about 10 μm, and as small as a few nm.
Liposomes consist basically of a phospholipid bilayer forming a shell around an aqueous core. Advantages include the lipophilicity of the outer layers which “mimic” the outer membrane layers of cells and that they are taken up relatively easily by a variety of cells. Polymeric vehicles typically consist of micro/nanospheres and micro/nanocapsules formed of biocompatible polymers, which are either biodegradable (for example, polylactic acid) or non-biodegradable (for example, ethylenevinyl acetate). Some of the advantages of the polymeric devices are ease of manufacture and high loading capacity, range of size from nanometer to micron diameter, as well as controlled release and degradation profile.
In some embodiments, the particles comprise an antigen-binding molecule on their surface, which is immuno-interactive with a marker that is expressed at higher levels on antigen-presenting cells (e.g., dendritic cells) than on non-antigen-presenting cells. Illustrative markers of this type include MGL, DCL-1, DEC-205, macrophage mannose R, DC-SIGN or other DC or myeloid specific (lectin) receptors, as for example disclosed by Hawiger et al. (2001, J Exp Med 194:769), Kato et al. 2003, J Biol Chem 278:34035), Benito et al. (2004, J Am Chem Soc 126:10355), Schjetne, et al. (2002, Int Immunol 14:1423) and van Vliet et al., 2006, Nat Immunol 7(11):1200-1208; Immunobiology 211:577-585).
The immunomodulating particles of the present invention can be prepared from a combination of the bioactive agent(s), and a surfactant, excipient or polymeric material. In some embodiments, the particles are biodegradable and biocompatible, and optionally are capable of biodegrading at a controlled rate for delivery of a therapeutic or diagnostic agent. The particles can be made of a variety of materials. Both inorganic and organic materials can be used. Polymeric and non-polymeric materials, such as fatty acids, may be used. Other suitable materials include, but are not limited to, gelatine, polyethylene glycol, trehalulose, dextran and chitosan. Particles with degradation and release times ranging from seconds to months can be designed and fabricated, based on factors such as the particle material.
3.2.6.1 Polymeric ParticlesPolymeric particles may be formed from any biocompatible and desirably biodegradable polymer, copolymer, or blend. The polymers may be tailored to optimize different characteristics of the particle including: i) interactions between the bioactive agents to be delivered and the polymer to provide stabilization of the bioactive agents and retention of activity upon delivery; ii) rate of polymer degradation and, thereby, rate of agent release profiles; iii) surface characteristics and targeting capabilities via chemical modification; and iv) particle porosity.
Surface eroding polymers such as polyanhydrides may be used to form the particles. For example, polyanhydrides such as poly[(p-carboxyphenoxy)-hexane anhydride] (PCPH) may be used. Biodegradable polyanhydrides are described in U.S. Pat. No. 4,857,311.
In other embodiments, bulk-eroding polymers such as those based on polyesters including poly(hydroxy acids) or poly(esters) can be used. For example, polyglycolic acid (PGA), polylactic acid (PLA), or copolymers thereof may be used to form the particles. The polyester may also have a charged or functionalizable group, such as an amino acid. In illustrative examples, particles with controlled release properties can be formed of poly(D,L-lactic acid) and/or poly(D,L-lactic-co-glycolic acid) (“PLGA”), which incorporate a surfactant such as DPPC.
Other polymers include poly(alkylcyanoacrylates), polyamides, polycarbonates, polyalkylenes such as polyethylene, polypropylene, poly(ethylene glycol), poly(ethylene oxide), poly(ethylene terephthalate), poly vinyl compounds such as polyvinyl alcohols, polyvinyl ethers, and polyvinyl esters, polymers of acrylic and methacrylic acids, celluloses and other polysaccharides, and peptides or proteins, or copolymers or blends thereof. Polymers may be selected with or modified to have the appropriate stability and degradation rates in vivo for different controlled drug delivery applications.
In some embodiments, particles are formed from functionalized polyester graft copolymers, as described in Hrkach et al. (1995, Macromolecules, 28:4736-4739; and “Poly(L-Lactic acid-co-amino acid) Graft Copolymers: A Class of Functional Biodegradable Biomaterials” in Hydrogels and Biodegradable Polymers for Bioapplications, ACS Symposium Series No. 627, Raphael M. Ottenbrite et al., Eds., American Chemical Society, Chapter 8, pp. 93-101, 1996.)
Materials other than biodegradable polymers may be used to form the particles. Suitable materials include various non-biodegradable polymers and various excipients. The particles also may be formed of the bioactive agent(s) and surfactant alone.
Polymeric particles may be prepared using single and double emulsion solvent evaporation, spray drying, solvent extraction, solvent evaporation, phase separation, simple and complex coacervation, interfacial polymerization, and other methods well known to those of ordinary skill in the art. Particles may be made using methods for making microspheres or microcapsules known in the art, provided that the conditions are optimized for forming particles with the desired diameter.
Methods developed for making microspheres for delivery of encapsulated agents are described in the literature, for example, as described in Doubrow, M., Ed., “Microcapsules and Nanoparticles in Medicine and Pharmacy,” CRC Press, Boca Raton, 1992. Methods also are described in Mathiowitz and Langer (1987, J. Controlled Release 5:13-22); Mathiowitz et al. (1987, Reactive Polymers 6:275-283); and Mathiowitz et al. (1988, J. Appl. Polymer Sci. 3: 755-774) as well as in U.S. Pat. No. 5,213,812, U.S. Pat. No. 5,417,986, U.S. Pat. No. 5,360,610, and U.S. Pat. No. 5,384,133. The selection of the method depends on the polymer selection, the size, external morphology, and crystallinity that is desired, as described, for example, by Mathiowitz et al. (1990, Scanning Microscopy 4:329-340; 1992, J. Appl. Polymer Sci. 45:125-134); and Benita et al. (1984, J. Pharm. Sci. 73:1721-1724).
In solvent evaporation, described for example, in Mathiowitz et al., (1990), Benita; and U.S. Pat. No. 4,272,398 to Jaffe, the polymer is dissolved in a volatile organic solvent, such as methylene chloride. Several different polymer concentrations can be used, for example, between 0.05 and 2.0 g/mL The bioactive agent(s), either in soluble form or dispersed as fine particles, is (are) added to the polymer solution, and the mixture is suspended in an aqueous phase that contains a surface-active agent such as poly(vinyl alcohol). The aqueous phase may be, for example, a concentration of 1% poly(vinyl alcohol) w/v in distilled water. The resulting emulsion is stirred until most of the organic solvent evaporates, leaving solid microspheres, which may be washed with water and dried overnight in a lyophilizer Microspheres with different sizes (between 1 and 1000 μm) and morphologies can be obtained by this method.
Solvent removal was primarily designed for use with less stable polymers, such as the polyanhydrides. In this method, the agent is dispersed or dissolved in a solution of a selected polymer in a volatile organic solvent like methylene chloride. The mixture is then suspended in oil, such as silicon oil, by stirring, to form an emulsion. Within 24 hours, the solvent diffuses into the oil phase and the emulsion droplets harden into solid polymer microspheres Unlike the hot-melt microencapsulation method described for example in Mathiowitz et al. (1987, Reactive Polymers 6:275), this method can be used to make microspheres from polymers with high melting points and a wide range of molecular weights. Microspheres having a diameter for example between one and 300 microns can be obtained with this procedure.
With some polymeric systems, polymeric particles prepared using a single or double emulsion technique, vary in size depending on the size of the droplets. If droplets in water-in-oil emulsions are not of a suitably small size to form particles with the desired size range, smaller droplets can be prepared, for example, by sonication or homogenation of the emulsion, or by the addition of surfactants.
If the particles prepared by any of the above methods have a size range outside of the desired range, particles can be sized, for example, using a sieve, and further separated according to density using techniques known to those of skill in the art.
The polymeric particles can be prepared by spray drying. Methods of spray drying, such as that disclosed in WO 96/09814 by Sutton and Johnson, disclose the preparation of smooth, spherical microparticles of a water-soluble material with at least 90% of the particles possessing a mean size between 1 and 10 μm.
3.2.6.2 LiposomesLiposomes can be produced by standard methods such as those reported by Kim et al. (1983, Biochim. Biophys. Acta 728:339-348); Liu et al. (1992, Biochim. Biophys. Acta 1104:95-101); Lee et al. (1992, Biochim. Biophys. Acta. 1103:185-197), Brey et al. (U.S. Pat. Appl. Pub. 20020041861), Hass et al. (U.S. Pat. Appl. Pub. 20050232984), Kisak et al. (U.S. Pat. Appl. Pub. 20050260260) and Smyth-Templeton et al. (U.S. Pat. Appl. Pub. 20060204566). Additionally, reference may be made to Copeland et al. (2005, Immunol. Cell Biol. 83:95-105) who review lipid based particulate formulations for the delivery of antigen, and to Bramwell et al. (2005, Crit Rev Ther Drug Carrier Syst. 22(2):151-214; 2006, J Pharm Pharmacol. 58(6):717-728) who review particulate delivery systems for vaccines, including methods for the preparation of protein-loaded liposomes. Many liposome formulations using a variety of different lipid components have been used in various in vitro cell culture and animal experiments. Parameters have been identified that determine liposomal properties and are reported in the literature, for example, by Lee et al. (1992, Biochim. Biophys. Acta. 1103:185-197); Liu et al. (1992, Biochim. Biophys. Acta. 1104:95-101); and Wang et al. (1989, Biochem. 28:9508-9510).
Briefly, the lipids of choice (and any organic-soluble bioactive), dissolved in an organic solvent, are mixed and dried onto the bottom of a glass tube under vacuum. The lipid film is rehydrated using an aqueous buffered solution containing any water-soluble bioactives to be encapsulated by gentle swirling. The hydrated lipid vesicles can then be further processed by extrusion, submitted to a series of freeze-thawing cycles or dehydrated and then rehydrated to promote encapsulation of bioactives. Liposomes can then be washed by centrifugation or loaded onto a size-exclusion column to remove unentrapped bioactive from the liposome formulation and stored at 4° C. The basic method for liposome preparation is described in more detail in Thierry et al. (1992, Nuc. Acids Res. 20:5691-5698).
A particle carrying a payload of bioactive agent(s) can be made using the procedure as described in: Pautot et al. (2003, Proc. Natl. Acad. Sci. USA 100(19): 10718-21). Using the Pautot et al. technique, streptavidin-coated lipids (DPPC, DSPC, and similar lipids) can be used to manufacture liposomes. The drug encapsulation technique described by Needham et al. (2001, Advanced Drug Delivery Reviews 53(3):285-305) can be used to load these vesicles with one or more active agents.
The liposomes can be prepared by exposing chloroformic solution of various lipid mixtures to high vacuum and subsequently hydrating the resulting lipid films (DSPC/CHOL) with pH 4 buffers, and extruding them through polycarbonated filters, after a freezing and thawing procedure. It is possible to use DPPC supplemented with DSPC or cholesterol to increase encapsulation efficiency or increase stability, etc. A transmembrane pH gradient is created by adjusting the pH of the extravesicular medium to 7.5 by addition of an alkalinization agent. A bioactive agent (e.g., small molecule NF-κB inhibitors, mTOR inhibitors or Syk inhibitors, which are, for example, weak bases) can be subsequently entrapped by addition of a solution of the bioactive agent in small aliquots to the vesicle solution, at an elevated temperature, to allow accumulation of the bioactive agent inside the liposomes.
Other lipid-based particles suitable for the delivery of the bioactive agents of the present invention such as niosomes are described by Copeland et al. (2005, Immunol. Cell Biol. 83:95-105).
3.2.6.3 Ceramic ParticlesCeramic particles may also be used to deliver the bioactive agents of the invention. These particles are typically prepared using processes similar to the well known sol-gel process and usually require simple and room temperature conditions as described for example in Brinker et al. (“Sol-Gel Science: The Physics and Chemistry of Sol-Gel Processing;” Academic Press: San Diego, 1990, p-60), and Avnir et al. (1994, Chem. Mater. 6, 1605). Ceramic particles can be prepared with desired size, shape and porosity, and are extremely stable. They also effectively protect doped molecules (polypeptides, drugs etc.) against denaturation induced by extreme pH and temperature (Jain et al., 1998. J. Am. Chem. Soc. 120:11092-11095). In addition, their surfaces can be easily functionalized with different groups (Lal et al., 2000. Chem. Mater. 12:2632-2639; Badley et al., 1990, Langmuir 6:792-801), and therefore they can be attached to a variety of monoclonal antibodies and other ligands in order to target them to desired sites in vivo.
Various ceramic particles have been described for delivery in vivo of active agent-containing payloads. For example, British Patent 1 590 574 discloses incorporation of biologically active components in a sol-gel matrix. International Publication WO 97/45367 discloses controllably dissolvable silica xerogels prepared via a sol-gel process, into which a biologically active agent is incorporated by impregnation into pre-sintered particles (1 to 500 μm) or disks. International Publication WO 0050349 discloses controllably biodegradable silica fibers prepared via a sol-gel process, into which a biologically active agent is incorporated during synthesis of the fiber. U.S. Pat. Appl. Pub. 20040180096 describes ceramic nanoparticles in which a bioactive substance is entrapped. The ceramic nanoparticles are made by formation of a micellar composition of the dye. The ceramic material is added to the micellar composition and the ceramic nanoparticles are precipitated by alkaline hydrolysis. U.S. Pat. Appl. Pub. 20050123611 discloses controlled release ceramic particles comprising an active material substantially homogeneously dispersed throughout the particles. These particles are prepared by mixing a surfactant with an apolar solvent to prepare a reverse micelle solution; (b) dissolving a gel precursor, a catalyst, a condensing agent and a soluble active material in a polar solvent to prepare a precursor solution; (c) combining the reverse micelle solution and the precursor solution to provide an emulsion and (d) condensing the precursor in the emulsion. U.S. Pat. Appl. Pub. 20060210634 discloses adsorbing bioactive substances onto ceramic particles comprising a metal oxide (e.g., titanium oxide, zirconium oxide, scandium oxide, cerium oxide and yttrium oxide) by evaporation. Kortesuo et al. (2000, Int J Pharm. 200(2):223-229) disclose a spray drying method to produce spherical silica gel particles with a narrow particle size range for controlled delivery of drugs such as toremifene citrate and dexmedetomidine HCl. Wang et al. (2006, Int J Pharm. 308(1-2):160-167) describe the combination of adsorption by porous CaCO3 microparticles and encapsulation by polyelectrolyte multilayer films for delivery of bioactive substances.
3.2.6.4 Ballistic ParticlesThe bioactive agents of the present invention may be attached to (e.g., by coating or conjugation) or otherwise associated with particles suitable for use in needleless or “ballistic” (biolistic) delivery. Illustrative particles for ballistic delivery are described, for example, in: WO 02/101412; WO 02/100380; WO 02/43774; WO 02/19989; WO 01/93829; WO 01/83528; WO 00/63385; WO 00/26385; WO 00/19982; WO 99/01168; WO 98/10750; and WO 97/48485. It shall be understood, however, that such particles are not limited to their use with a ballistic delivery device and can otherwise be administered by any alternative technique (e.g., injection or microneedle delivery) through which particles are deliverable to immune cells.
The bioactive agents can be coated or chemically coupled to carrier particles (e.g., core carriers) using a variety of techniques known in the art. Carrier particles are selected from materials that have a suitable density in the range of particle sizes typically used for intracellular delivery. The optimum carrier particle size will, of course, depend on the diameter of the target cells. Illustrative particles have a size ranging from about 0.01 to about 250 μm, from about 10 to about 150 μm, and from about 20 to about 60 μm; and a particle density ranging from about 0.1 to about 25 g/cm3, and a bulk density of about 0.5 to about 3.0 g/cm3, or greater. Non-limiting particles of this type include metal particles such as, tungsten, gold, platinum and iridium carrier particles. Tungsten particles are readily available in average sizes of 0.5 to 2.0 μm in diameter. Gold particles or microcrystalline gold (e.g., gold powder A1570, available from Engelhard Corp., East Newark, N.J.) may also be used. Gold particles provide uniformity in size (available from Alpha Chemicals in particle sizes of 1-3 μm, or available from Degussa, South Plainfield, N.J. in a range of particle sizes including 0.95 μm) and low toxicity. Microcrystalline gold provides a diverse particle size distribution, typically in the range of 0.1-5 μm. The irregular surface area of microcrystalline gold provides for highly efficient coating with the active agents of the present invention.
Many methods are known and have been described for adsorbing, coupling or otherwise attaching bioactive molecules (e.g., hydrophilic molecules such as proteins and nucleic acids) onto particles such as gold or tungsten particles. In illustrative examples, such methods combine a predetermined amount of gold or tungsten with the bioactive molecules, CaCl2 and spermidine. In other examples, ethanol is used to precipitate the bioactive molecules onto gold or tungsten particles (see, for example, Jumar et al., 2004, Phys Med. Biol. 49:3603-3612). The resulting solution is suitably vortexed continually during the coating procedure to ensure uniformity of the reaction mixture. After attachment of the bioactive molecules, the particles can be transferred for example to suitable membranes and allowed to dry prior to use, coated onto surfaces of a sample module or cassette, or loaded into a delivery cassette for use in particular particle-mediated delivery instruments.
The formulated compositions may suitably be prepared as particles using standard techniques, such as by simple evaporation (air drying), vacuum drying, spray drying, freeze drying (lyophilization), spray-freeze drying, spray coating, precipitation, supercritical fluid particle formation, and the like. If desired, the resultant particles can be dandified using the techniques described in International Publication WO 97/48485.
3.2.6.5 SurfactantsSurfactants, which can be incorporated into particles, include phosphoglycerides. Exemplary phosphoglycerides include phosphatidylcholines, such as the naturally occurring surfactant, L-α-phosphatidylcholine dipalmitoyl (“DPPC”). The surfactants advantageously improve surface properties by, for example, reducing particle-particle interactions, and can render the surface of the particles less adhesive. The use of surfactants endogenous to the lung may avoid the need for the use of non-physiologic surfactants.
Providing a surfactant on the surfaces of the particles can reduce the tendency of the particles to agglomerate due to interactions such as electrostatic interactions, Van der Waals forces, and capillary action. The presence of the surfactant on the particle surface can provide increased surface rugosity (roughness), thereby improving aerosolization by reducing the surface area available for intimate particle-particle interaction.
Surfactants known in the art can be used including any naturally occurring surfactant. Other exemplary surfactants include diphosphatidyl glycerol (DPPG); hexadecanol; fatty alcohols such as polyethylene glycol (PEG); polyoxyethylene-9-lauryl ether; a surface active fatty acid, such as palmitic acid or oleic acid; sorbitan trioleate (Span 85); glycocholate; surfactin; a poloxamer; a sorbitan fatty acid ester such as sorbitan trioleate; tyloxapol and a phospholipid.
The particles may be used as vehicles for delivery of inhibitor and aggrecan antigen to antigen-presenting cells ex vivo. However, in advantageous embodiments, the particles are used to deliver the bioactive compounds to antigen-presenting cells in vivo, as disclosed for example in U.S. Pat. Appl. Pub. 20100151000, which is hereby incorporated by reference herein in its entirety.
3.2.7 Ex Vivo Antigen-Presenting Cell EmbodimentsInhibitor and aggrecan antigen can be delivered ex vivo into antigen-presenting cells in various forms, and may be soluble or particulate in nature. The antigen-presenting cells or their precursors may be obtained from the subject to be treated (i.e., autologous antigen-presenting cells or precursors). Alternatively, the antigen-presenting cells or their precursors are obtained or derived from a donor that is MHC-matched or mismatched with the subject (i.e., an allogeneic antigen-presenting cell). Suitably, in these embodiments, the donor is histocompatible with the subject.
In the above embodiments, the antigen-presenting cells are contacted with one or more inhibitors as described for example in Sections 3.2.3, 3.2.4 and 3.2.5, or with a polynucleotide from which the inhibitor is expressible, for a time and under conditions sufficient to inhibit, reduce or otherwise impair NF-κB activity and/or mTOR activity and/or Syk activity in the antigen-presenting cell. The amount of soluble or particulate inhibitor to be placed in contact with antigen-presenting cells or their precursors can be determined empirically by routine methods known to persons of skill in the art. In some advantageous embodiments the antigen-presenting cells are incubated in the presence of inhibitor for at least about 2, 3, 4, 5, 6, 7, 8, 12, 24, 36, 48, 60, 72, 84, 96 hours, or for less than about 96, 84, 72, 60, 48, 36, 24, 12, 8, 7, 6, 5, 4, 3 or 2 hours. In specific embodiments, antigen-presenting cells are incubated with inhibitor (e.g., a NF-κB inhibitor or mTOR inhibitor or Syk inhibitor) for about 48 to about 60 h at 35° C.-38° C. or for as much time as required to inhibit, reduce or otherwise impair the activity of NF-κB, mTOR or Syk. In illustrative examples of this type, the antigen-presenting cells may be incubated in the presence of an activator of antigen-presenting cells such as lipopolysaccharide (LPS) and optionally additional inhibitors (e.g., additional NF-κB inhibitors such as Vitamin D and dexamethasone).
The antigen-presenting cells or their precursors are also contacted with aggrecan antigen as described for example in Section 3.2.1, or with aggrecan APL as described for example in Section 3.2.2, or with a polynucleotide from which the antigen or APL is expressible, for a time and under conditions sufficient for the antigen or a processed form thereof to be presented by the antigen-presenting cells. Suitably, the inhibitor and/or the antigen or a polynucleotide from which they are expressible are in soluble form or in particulate form as described for example in Section 3.2.6. The amount of soluble or particulate antigen or APL to be placed in contact with antigen-presenting cells or their precursors can be determined empirically by routine methods known to persons of skill in the art. In some advantageous embodiments the antigen-presenting cells are incubated in the presence of aggrecan antigen for at least about 2, 3, 4, 5, 6, 7, 8, 12, 24, 36, 48, 60, 72, 84, 96 hours, or for less than about 96, 84, 72, 60, 48, 36, 24, 12, 8, 7, 6, 5, 4, 3 or 2 hours or even for less that about 60, 50, 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3 or 2 minutes. The time and dose of polypeptide or peptides necessary for the cells to optionally process and present aggrecan antigen may be determined using pulse-chase protocols in which exposure to aggrecan antigen is followed by a washout period and exposure to a read-out system e.g., ability to elicit suppression or inhibition of an aggrecan antigen-specific effector lymphocyte response. Once the optimal time and dose necessary for cells to express the aggrecan antigen or processed form thereof on their surface is determined, a protocol may be used to prepare cells and aggrecan antigen for inducing tolerogenic responses. Those of skill in the art will recognize in this regard that the length of time necessary for an antigen-presenting cell to present an antigen on its surface may vary depending on the antigen or form of antigen employed, its dose, and the antigen-presenting cell employed, as well as the conditions under which antigen loading is undertaken. These parameters can be determined by the skilled artisan using routine procedures.
In some embodiments, antigen-presenting cells are incubated with antigen or APL for about 1 to 6 h at 37° C., although it is also possible to expose antigen-presenting cells to antigen for the duration of incubation with growth factors and inhibitor. Usually, for purified antigens and peptides, 0.1-10 μg/mL is suitable for producing antigen-specific antigen-presenting cells. In illustrative examples, presentation of peptide antigen can be achieved using much shorter periods of incubation (e.g., about 5, 10, 15, 20, 30, 40, 50 minutes) using antigen at a concentration of about 10-20 μg/mL.
3.2.7.1 Sources of Antigen-Presenting Cells and their PrecursorsAntigen-presenting cells or their precursors can be isolated by methods known to those of skill in the art. The source of such cells will differ depending upon the antigen-presenting cell required for modulating a specified immune response. In this context, the antigen-presenting cell can be selected from dendritic cells, macrophages, monocytes and other cells of myeloid lineage.
Typically, precursors of antigen-presenting cells can be isolated from any tissue, but are most easily isolated from blood, cord blood or bone marrow (Sorg et al., 2001. Exp Hematol 29:1289-1294; Zheng et al., 2000. J Hematother Stem Cell Res 9:453-464). It is also possible to obtain suitable precursors from diseased tissues such as rheumatoid synovial tissue or fluid following biopsy or joint tap (Thomas et al., 1994a, J Immunol 153:4016-4028; Thomas et al., 1994b, Arthritis Rheum 37(4)). Other examples include, but are not limited to liver, spleen, heart, kidney, gut and tonsil (Lu et al., 1994. J Exp Med 179:1823-1834; McIlroy et al., 2001. Blood 97:3470-3477; Vremec et al., 2000. J Immunol 159:565-573; Hart and Fabre, 1981. J Exp Med 154(2):347-361; Hart and McKenzie, 1988. J Exp Med 168(1):157-170; Pavli et al., 1990. Immunology 70(1):40-47).
Leukocytes isolated directly from tissue provide a major source of antigen-presenting cell precursors. Typically, these precursors can only differentiate into antigen-presenting cells by culturing in the presence or absence of various growth factors. According to the practice of the present invention, the antigen-presenting cells may be so differentiated from crude mixtures or from partially or substantially purified preparations of precursors. Leukocytes can be conveniently purified from blood or bone marrow by density gradient centrifugation using, for example, Ficoll Hypaque which eliminates neutrophils and red cells (peripheral blood mononuclear cells or PBMCs), or by ammonium chloride lysis of red cells (leukocytes or white blood cells). Many precursors of antigen-presenting cells are present in peripheral blood as non-proliferating monocytes, which can be differentiated into specific antigen-presenting cells, including macrophages and dendritic cells, by culturing in the presence of specific cytokines.
Tissue-derived precursors such as precursors of tissue dendritic cells or of Langerhans cells are typically obtained by mincing tissue (e.g., basal layer of epidermis) and digesting it with collagenase or dispase followed by density gradient separation, or selection of precursors based on their expression of cell surface markers. For example, Langerhans cell precursors express CD1 molecules as well as HLA-DR and can be purified on this basis.
In some embodiments, the antigen-presenting cell precursor is a precursor of macrophages. Generally these precursors can be obtained from monocytes of any source and can be differentiated into macrophages by prolonged incubation in the presence of medium and macrophage colony stimulating factor (M-CSF) (Erickson-Miller et al., 1990. Int J Cell Cloning 8:346-356; Metcalf and Burgess, 1982. J Cell Physiol 111:275-283).
In other embodiments, the antigen presenting cell precursor is a precursor of Langerhans cells. Usually, Langerhans cells can be generated from human monocytes or CD34+ bone marrow precursors in the presence of granulocyte/macrophage colony-stimulating factor (GM-CSF), IL-4/TNFα and TGFβ (Geissmann et al., 1998. J Exp Med 187:961-966; Strobl et al., 1997a. Blood 90:1425-1434; Strobl et al., 1997b. dv Exp Med Biol 417:161-165; Strobl et al., 1996. J Immunol 157:1499-1507).
In still other embodiments, the antigen-presenting cell precursor is a precursor of dendritic cells. Several potential dendritic cell precursors can be obtained from peripheral blood, cord blood or bone marrow. These include monocytes, CD34+ stem cells, granulocytes, CD33+CD11c+ DC precursors, and committed myeloid progenitors—described below.
Monocytes:
Monocytes can be purified by adherence to plastic for 1-2 h in the presence of tissue culture medium (e.g., RPMI) and serum (e.g., human or fetal calf serum), or in serum-free medium (Anton et al., 1998. Scand J Immunol 47:116-121; Araki et al., 2001. Br J Haematol 114:681-689; Mackensen et al., 2000. Int J Cancer 86:385-392; Nestle et al., 1998. Nat Med 4:328-332; Romani et al., 1996. J Immunol Meth 196:137-151; Thurner et al., 1999. J Immunol Methods 223:1-15). Monocytes can also be elutriated from peripheral blood (Garderet et al., 2001. J Hematother Stem Cell Res 10:553-567). Monocytes can also be purified by immunoaffinity techniques, including immunomagnetic selection, flow cytometric sorting or panning (Araki et al., 2001, supra; Battye and Shortman, 1991. Curr. Opin. Immunol. 3:238-241), with anti-CD14 antibodies to obtain CD14hi cells. The numbers (and therefore yield) of circulating monocytes can be enhanced by the in vivo use of various cytokines including GM-CSF (Groopman et al., 1987. N Engl J Med 317:593-598; Hill et al., 1995. J Leukoc Biol 58:634-642). Monocytes can be differentiated into dendritic cells by prolonged incubation in the presence of GM-CSF and IL-4 (Romani et al., 1994. J Exp Med 180, 83-93; Romani et al., 1996, supra). A combination of GM-CSF and IL-4 at a concentration of each at between about 200 to about 2000 U/mL, more preferably between about 500 to about 1000 U/mL and even more preferably between about 800 U/mL (GM-CSF) and 1000 U/mL (IL-4) produces significant quantities of immature dendritic cells, i.e., antigen-capturing phagocytic dendritic cells. Other cytokines which promote differentiation of monocytes into antigen-capturing phagocytic dendritic cells include, for example, IL-13.
CD34+ Stem Cells:
Dendritic cells can also be generated from CD34+ bone marrow derived precursors in the presence of GM-CSF, TNFα±stem cell factor (SCF, c-kitL), or GM-CSF, IL-4±flt3L (Bai et al., 2002. Int J Oncol 20:247-53; Chen et al., 2001. Clin Immunol 98:280-292; Loudovaris et al., 2001. J Hematother Stem Cell Res 10:569-578). CD34+ cells can be derived from a bone marrow aspirate or from blood and can be enriched as for monocytes using, for example, immunomagnetic selection or immunocolumns (Davis et al., 1994. J Immunol Meth 175:247-257). The proportion of CD34+ cells in blood can be enhanced by the in vivo use of various cytokines including (most commonly) G-CSF, but also flt3L and progenipoietin (Fleming et al., 2001. Exp Hematol 29:943-951; Pulendran et al., 2000. J Immunol 165:566-572; Robinson et al., 2000. J Hematother Stem Cell Res 9:711-720).
Other Myeloid Progenitors:
DC can be generated from committed early myeloid progenitors in a similar fashion to CD34+ stem cells, in the presence of GM-CSF and IL-4/TNF. Such myeloid precursors infiltrate many tissues in inflammation, including rheumatoid arthritis synovial fluid (Santiago-Schwarz et al., 2001. J Immunol. 167:1758-1768). Expansion of total body myeloid cells including circulating dendritic cell precursors and monocytes, can be achieved with certain cytokines, including flt-3 ligand, granulocyte colony-stimulating factor (G-CSF) or progenipoietin (pro-GP) (Fleming et al., 2001, supra; Pulendran et al., 2000, supra; Robinson et al., 2000, supra). Administration of such cytokines for several days to a human or other mammal would enable much larger numbers of precursors to be derived from peripheral blood or bone marrow for in vitro manipulation. Dendritic cells can also be generated from peripheral blood neutrophil precursors in the presence of GM-CSF, IL-4 and TNFα (Kelly et al., 2001. Cell Mol Biol (Noisy-le-grand) 47, 43-54; Oehler et al., 1998. J Exp Med. 187:1019-1028). It should be noted that dendritic cells can also be generated, using similar methods, from acute myeloid leukemia cells (Oehler et al., 2000. Ann Hematol. 79, 355-62).
Tissue DC Precursors and Other Sources of APC Precursors:
Other methods for DC generation exist from, for example, thymic precursors in the presence of IL-3+/−GM-CSF, and liver DC precursors in the presence of GM-CSF and a collagen matrix. Transformed or immortalized dendritic cell lines may be produced using oncogenes such as v-myc as for example described by (Paglia et al., 1993, supra) or by myb (Banyer and Hapel, 1999, supra; Gonda et al., 1993, supra).
Circulating DC Precursors:
These have been described in human and mouse peripheral blood. One can also take advantage of particular cell surface markers for identifying suitable dendritic cell precursors. Specifically, various populations of dendritic cell precursors can be identified in blood by the expression of CD11c and the absence or low expression of CD14, CD19, CD56 and CD3 (O'Doherty et al., 1994. Immunology 82, 487-493; O'Doherty et al., 1993. J Exp Med 178:1067-1078). These cells can also be identified by the cell surface markers CD13 and CD33 (Thomas et al., 1993b. J Immunol 151(12):6840-6852). A second subset, which lacks CD14, CD19, CD56 and CD3, known as plasmacytoid dendritic cell precursors, does not express CD11c, but does express CD123 (IL-3R chain) and HLA-DR (Farkas et al., 2001. Am J Pathol 159. 237-243; Grouard et al., 1997. J Exp Med 185:1101-1111; Rissoan et al., 1999. Science 283:1183-1186). Most circulating CD11c+ dendritic cell precursors are HLA-DR+, however some precursors may be HLA-DR-. The lack of MHC class II expression has been clearly demonstrated for peripheral blood dendritic cell precursors (del Hoyo et al., 2002. Nature 415:1043-1047).
Optionally, CD33+CD14−/lo or CD11c+HLA-DR+, lineage marker-negative dendritic cell precursors described above can be differentiated into more mature antigen-presenting cells by incubation for 18-36 h in culture medium or in monocyte conditioned medium (Thomas et al., 1993b, supra; Thomas and Lipsky, 1994. J Immunol 153:4016-4028) (O'Doherty et al., 1993, supra). Alternatively, following incubation of peripheral blood non-T cells or unpurified PBMC, the mature peripheral blood dendritic cells are characterized by low density and so can be purified on density gradients, including metrizamide and Nycodenz (Freudenthal and Steinman, 1990. Proc Natl Acad Sci USA 87:7698-7702; Vremec and Shortman, 1997. J Immunol 159:565-573), or by specific monoclonal antibodies, such as but not limited to the CMRF-44 mAb (Fearnley et al., 1999. Blood 93:728-736; Vuckovic et al., 1998. Exp Hematol 26:1255-1264). Plasmacytoid dendritic cells can be purified directly from peripheral blood on the basis of cell surface markers, and then incubated in the presence of IL-3 (Grouard et al., 1997, supra; Rissoan et al., 1999, supra). Alternatively, plasmacytoid DC can be derived from density gradients or CMRF-44 selection of incubated peripheral blood cells as above.
In general, for dendritic cells generated from any precursor, when incubated in the presence of activation factors such as monocyte-derived cytokines, lipopolysaccharide and DNA containing CpG repeats, cytokines such as TNFα, IL-6, IFN-α, IL-1β, necrotic cells, re-adherence, whole bacteria, membrane components, RNA or polyIC, immature dendritic cells will become activated (Clark, 2002. J Leukoc Biol 71:388-400; Hacker et al., 2002. Immunology 105:245-251; Kaisho and Akira, 2002. Biochim Biophys Acta 1589:1-13; Koski et al., 2001. Crit Rev Immunol 21:179-189). This process of dendritic cell activation is inhibited in the presence of NF-κB inhibitors (O'Sullivan and Thomas, 2002. J Immunol 168:5491-5498).
3.2.8 In Vivo Antigen-Presenting Cell EmbodimentsIn other embodiments, the number of aggrecan-specific tolerogenic antigen-presenting cells in the subject is increased by co-administering to the subject an NF-κB inhibitor and/or a mTOR inhibitor, and/or a Syk inhibitor (sometimes collectively referred to herein as “inhibitor(s)”), together with an aggrecan antigen that corresponds in whole, or in part, to an aggrecan polypeptide, including citrullinated forms thereof. The inhibitor(s) and/or the antigen may be in nucleic acid form (i.e., in which they are produced from a nucleic acid construct) or in non-nucleic acid form. The inhibitor(s) and the antigen may be co-administered in soluble or in particulate form (e.g., the antigen and the inhibitor are both in soluble form, or one of the antigen or inhibitor is in soluble form and the other is in particulate form, or the antigen and the inhibitor are both in particulate form). In specific embodiments, both the inhibitor(s) and the antigen are co-administered in particulate form. Desirably, the inhibitor(s) and the antigen are contained in the same particle. Once administered, the particles are taken up by antigen-presenting cells in the subject (e.g., by phagocytosis or endocytosis), and the inhibitor(s)/antigen payload is released into the interior of the cell.
3.2.9 Ex Vivo Regulatory Lymphocyte EmbodimentsAggrecan-specific antigen-presenting cells obtained or prepared ex vivo according to Section 3.2.7 are useful for modulating other immune cells, including T lymphocytes and B lymphocytes, and particularly for producing T lymphocytes and B lymphocytes that exhibit tolerance or anergy to aggrecan antigen (including citrullinated forms thereof). The efficiency of inducing lymphocytes, especially T lymphocytes, to exhibit tolerance/anergy for aggrecan antigen can be determined by assaying immune responses to that antigen including, but not limited to, assaying T lymphocyte cytolytic activity in vitro using for example the aggrecan-specific antigen-presenting cells as targets of aggrecan-specific cytolytic T lymphocytes (CTL); assaying aggrecan-specific T lymphocyte proliferation and cytokine response; and assaying T regulatory suppressive function (see, e.g., Vollenweider and Groseurth, 1992, J. Immunol. Meth. 149:133-135), measuring B cell response to the antigen using, for example, ELISPOT assays, and ELISA assays; interrogating cytokine profiles; or measuring delayed-type hypersensitivity (DTH) responses by test of skin reactivity to the aggrecan antigen (see, e.g., Chang et al. (1993, Cancer Res. 53:1043-1050). Other methods known to practitioners in the art, which can detect the presence of antigen on the surface of antigen-presenting cells after exposure to the antigen, are also contemplated by the present invention.
The tolerance/anergy-eliciting antigen-presenting cells have the capacity to efficiently present the aggrecan antigen, or processed form thereof, on one or both of MHC class I molecules and MHC class II molecules. Accordingly, both CD4+ T helper lymphocytes and CTL may be rendered tolerant/anergic by the agg-tolAPC of the invention. Moreover, the agg-tolAPC of the present invention can be charged with multiple aggrecan antigens on multiple MHCs to yield polyclonal or oligoclonal tolerance/anergy of T lymphocytes.
Thus, the present invention also provides aggrecan-specific tolerant/anergic or regulatory B or T lymphocytes, especially T lymphocytes, which fail to respond in an antigen-specific fashion to representation of the aggrecan antigen or which actively regulate prior immune responses or subsequent priming to the aggrecan antigen. The regulation is generally long lived and is maintained, for example, for at least about 3 months, and suitably years.
In specific embodiments, aggrecan-specific regulatory T lymphocytes are produced by contacting an aggrecan-specific antigen-presenting cell as defined above with a population of T lymphocytes, which may be obtained from any suitable source such as spleen or tonsil/lymph nodes but is preferably obtained from peripheral blood. The T lymphocytes can be used as crude preparations or as partially purified or substantially purified preparations, which are suitably obtained using standard techniques as, for example, described in “Immunochemical Techniques, Part G: Separation and Characterization of Lymphoid Cells” (Meth. in Enzymol. 108, Edited by Di Sabato et al., 1984, Academic Press). This includes rosetting with sheep red blood cells, passage across columns of nylon wool or plastic adherence to deplete adherent cells, immunomagnetic or flow cytometric selection using appropriate monoclonal antibodies as described (Cavanagh et al., 1998. Blood 92(5)inhibitor 1598-1607; Thomas et al., 1993. J Immunol 150inhibitor 821-834).
The preparation of T lymphocytes is contacted with the aggrecan-specific antigen-presenting cells as described for example in Section 3.2.7 for an adequate period of time for inducing regulatory function in the T lymphocytes towards the antigen or antigens presented by those antigen-presenting cells. This period will usually be at least about 1 day, and up to about 5 days. Generally, the proliferation of regulatory T lymphocytes produced after this procedure is short-lived, depending on the IL-2 concentration in the culture. The regulatory lymphocytes so prepared will typically produce IL-10 and/or other regulatory cytokines in an antigen-specific manner.
In some embodiments, a population of aggrecan-specific antigen-presenting cell precursors is cultured in the presence of a heterogeneous population of T lymphocytes, which is suitably obtained from peripheral blood, and at least one inhibitor, as described for example in Sections 3.2.3, 3.2.4 and 3.2.5, together with an aggrecan antigen, or with a polynucleotide from which the aggrecan antigen is expressible. These cells are cultured for a period of time and under conditions sufficient for: (1) the precursors to differentiate into antigen-presenting cells; (2) the level and/or functional activity of NF-κB and/or mTOR and/or Syk in those antigen-presenting cells to be abrogated or otherwise reduced; (3) the aggrecan antigen, or processed form thereof, to be presented by the antigen-presenting cells; and (4) the antigen-presenting cells to induce a subpopulation of the T lymphocytes to exhibit regulatory function towards the aggrecan antigen, wherein the subpopulation is characterized by suppression of an aggrecan-specific T cell response. This can occur using Ficoll-purified PBMC plus aggrecan antigen plus inhibitor (e.g., NF-κB and/or mTOR and/or Syk inhibitor(s)) since such a preparation contains both antigen-presenting cell precursors (e.g., dendritic cell precursors) and T lymphocytes.
Suitably, the aggrecan-specific antigen-presenting cells so produced induce one or more types of antigen-specific regulatory lymphocytes, especially regulatory T lymphocytes (also referred to herein as “Treg”). Several populations or subsets of Treg have been described (Shevach, 2006. Immunity 25:195-201; Lee et al. 2011, Adv Immunol. 112:25-71; Sakaguchi, 2011. Methods Mol Biol. 707:3-17), including, for example, a naturally-occurring, distinct population of CD4+CD25+Foxp3+ Treg known as natural Treg (nTreg) develop in the thymus and are present in healthy individuals from birth. The specificity of the T cell receptor (TCR) of nTreg is mainly self-reactive. Additionally, a population of CD4+CD25+Foxp3+ Treg can be induced in vivo in the periphery under various conditions, such as during certain defined conditions of antigen presentation and cytokine stimulation, and can induce tolerance (reviewed in Roncarolo et al., 2007. Nat Rev Immunol. 7: 585-598). Additional subsets of inducible Treg have been reported, including Th3 cells and Tr1 cells, and CD4+CD25+ Treg. Th3 cells produce TGF β and variable amounts of IL-4 and IL-10 (Chen et al., 1994. Science 265(5176):1237-1240). Tr1 cells secrete IL-10 (Groux et al., 1997. Nature 389(6652):737-742). CD8+ Treg have also been reported, which express low levels of Foxp3 (see, for example, Smith et al., 2008. Trends Immunol. 29(7):337-342; Guillonneau et al., 2010. Curr Opin Organ Transplant 15:751-756; Filaci et al., 2011. Autoimmunity 44(1):51-57; Picarda et al., 2011. Immunotherapy 3(4 Suppl): 35-37).
Numerous methods for isolating and expanding Treg are known in the art and include for example those disclosed by Brusko et al. (2008. Immunological Reviews 223:371-390), Gregori et al. (2011. Methods Mol Biol. 677:31-46), Gregori et al. (2007. Methods Mol Biol. 380:83-105), Ménoret et al., 2011. Methods Mol Biol. 677: 63-83), Wang, L. (2010. Methods Mol Biol. 595:403-412) and Daniel et al. (2011. Methods Mol Biol. 707:173-185).
Thus, the present invention provides means to generate large quantities of antigen-specific lymphocytes by stimulating lymphocytes with aggrecan-specific antigen-presenting cells of the invention e.g., for at least about 3 days, suitably at least about 5 days.
Aggrecan-specific tolerance can also be achieved by expressing in B lymphocytes (also referred to herein as B cells) a nucleic acid molecule that encodes an aggrecan antigen, wherein the expression of the nucleic acid molecule leads to presentation of the antigen or processed form thereof on the surface of the B lymphocytes.
In specific embodiments, the nucleic acid molecule further encodes an immunoglobulin (e.g., IgG) or a fragment of an immunoglobulin (e.g., Fv, Fab, Fab′ and F(ab′)2 immunoglobulin fragments, immunoglobulin heavy chain etc.) fused directly or indirectly to the aggrecan antigen (e.g., adjacent to the C-terminus of the antigen). In illustrative examples of this type, immunoglobulins (e.g., IgG) are used as carriers for presentation of an aggrecan antigen to the immune system, wherein the aggrecan antigen is fused at the N-terminus of the immunoglobulin heavy chain scaffold to form a fusion protein. The fusion protein is produced in ‘activated’ B cells by transducing (e.g., by retroviral infection) hematopoietic cells (e.g., bone marrow-derived cells) or lymphoid cells (e.g., B cells including B cell blasts that have been stimulated with lipopolysaccharide) with a nucleic acid molecule from which the fusion protein is producible to thereby produce tolerogenic APC. The aggrecan antigen or processed form thereof is presented by host major histocompatibility complex (MHC) on these B cells leading to tolerance because of the presentation and long-term in vivo expression of immunoglobulin (e.g., IgG) fusion proteins. Suitably, these B cells express MHC class II and co-stimulatory molecules (e.g., B7.1 and B7.2), which helps to recruit and trigger regulatory T cells that express CD25 via binding to CTLA-4 Immunoglobulin fusions of this type are described for example in U.S. Pat. Appl. Pub. No. 2002/0048562, which is hereby incorporated by reference herein in its entirety.
In illustrative examples of this type, antigen-specific tolerance is induced using antigen-Ig fusion protein delivered via a retroviral vector in B cells as described for example by Scott and colleagues (Zambidis et al., 1996. Proc Natl Acad Sci USA 93(10):5019-5024; Kang et al., 1999. Proc Natl Acad Sci USA 96(15):8609-8614; Agarwal et al., 2000. Clin Invest 106(2):245-252; El-Amine et al., 2002. Int Immunol 14(7):761-766; Melo et al., 2002. J Immunol 168(9):4788-4795; Song et al., 2004. Gene Ther 11(20):1487-1496; Lei et al., 2005. Blood 105(12):4865-4870; Xu et al., 2006. Mol Ther 13(1):42-48; Satpute et al., 2007. Arthritis Rheum 56(5):1490-1496; Scott, 2010. Haemophilia 16(102):89-94).
This B cell-mediated gene therapy approach has been used successful in disease models such as experimental autoimmune uveitis (EAU) (Agarwal et al., 2000, supra), EAE [induced either by myelin basic protein (MBP) or by myelin oligodendrocyte glycoprotein (MOG)] (Melo et al., 2002, supra), and the non-obese diabetic (NOD) mouse model of diabetes (Melo et al., 2002, supra; Song et al., 2004, supra). This approach has also been successful in inducing tolerance to factor VIII inhibitors in hemophilia A (Lei et al., 2005, supra) and (in combination with bone marrow (BM) transplantation) in the treatment of EAE (Xu et al., 2006, supra). B cell-mediated gene therapy has also been used successfully in the adjuvant-induced arthritis (AA) model (Satpute et al., 2007, supra).
The fusion protein can be delivered by any suitable means including administering a nucleic acid molecule from which the fusion protein is producible to a subject in need thereof. In specific embodiments, the subject is administered hematopoietic or lymphoid cells, which are transduced with the nucleic acid molecule.
In some embodiments, the B lymphocytes expressing the aggrecan antigen-encoding nucleic acid molecule are regulatory B lymphocytes (also referred to herein as “Breg” cells) an include but are not limited to a CD1dhighCD5+ B cell subset that regulates T cell mediated inflammatory and immune responses through secretion of IL-10, as disclosed for example by Tedder in U.S. Pat. Appl. Pub. No. 2011/013566, which is hereby incorporated by reference herein in its entirety.
3.2.10 MHC-Peptide Complex EmbodimentsThe present invention also contemplates the use of major histocompatibility complex (MHC)-peptide complexes for eliciting aggrecan-specific tolerogenesis. These complexes generally comprise an aggrecan peptide (e.g., a cit-aggrecan peptide) and an isolated (e.g., soluble) MHC component having an antigen-binding site, wherein the antigen is associated with the antigen-binding site. MHC-peptide complexes effectively substitute for the antigen-presenting cell and cause non-responsiveness in autoreactive antigen-specific T-lymphocytes and other cells of the immune system. The MHC component can be either a Class I or a Class II molecule. If desired, the transmembrane and/or intracellular domains of MHC molecules can also be included. The association between the peptide antigen and the antigen binding sites of the MHC protein can be by covalent or by non-covalent bonding.
The MHC molecules may be purified using any suitable protocol, illustrative examples of which involve solubilization of cells (e.g., lymphocytes) by treatment with papain, by treatment with 3M KC1, or by treatment with detergent. In an illustrative protocol, detergent extraction of MHC (e.g., Class II) molecules from lymphocytes is used followed by affinity purification. Detergent can then be removed by dialysis or selective binding beads, e.g., Bio Beads. Alternatively, the amino acid sequence of each of a number of MHC class I and II molecules are known, and their corresponding coding sequences have been cloned, thus permitting recombinant production of the MHC proteins.
The aggrecan antigen is generally a peptide (e.g., about 8 to about 15 amino acids in length), which is predicted to by the MHC molecule using standard algorithms known in the art. Alternatively, peptides (e.g., about 8 to about 15 amino acids in length) corresponding to overlapping portions of the aggrecan amino acid sequence can be used. The peptide(s) may be non-citrullinated or citrullinated.
The elements of the complex can be associated by standard means known in the art. The antigenic peptides can be associated non-covalently with the pocket portion of the MHC protein by, for example, mixing the two components. They can also be covalently bound using standard procedures by, for example, photo affinity labeling, (see e.g., Hall et al., 1985. Biochemistry 24:5702-5711).
Complexes comprising transmembrane-containing MHC molecules are conveniently administered after being incorporated in lipid monolayers or bilayers. Typically liposomes are used for this purpose but any form of lipid membrane, such as planar lipid membranes or the cell membrane of a cell (e.g., a red blood cell) may be used. The complexes are also conveniently incorporated into micelles.
Liposomes can be prepared according to standard methods, as described for example in Section 3.2.6.2. However, if the transmembrane region is deleted, the complex can be administered in a manner conventionally used for peptide-containing pharmaceuticals.
Micelles are commonly used in the art to increase solubility of molecules having nonpolar regions and may be used advantageously to incorporate the MHC-peptide complexes using methods well known in the art (see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed. (1985), which is hereby incorporated herein by reference in its entirety). Generally, complex-containing micelles are prepared using standard surfactants or detergents.
Exemplary methods of producing MHC-peptide complexes are described for example in U.S. Pat. Appl. Pub. No. 2003/0068363, which is hereby incorporated by reference herein in its entirety.
3.2.11 Chimeric Constructs Based on the Ligand Epitope Antigen Presenting SystemThe present invention also contemplates the use of the Ligand Epitope Antigen Presenting System (L.E.A.P.S.) technology for eliciting aggrecan-specific tolerogenesis. This technology converts a small peptide containing an autoantigen into a tolerogen by attaching it to an immune or T cell binding ligand (I/TCBL) and presenting it to an immune cell (Cihakova et al., 2008. Int Immunopharmacol 8:624; Rosenthal et al., 1998. Vaccine 17:535-542; Goel et al., 2003. Vaccine 21:4410; Goel et al., 2005. Front Biosci 966; Zimmerman et al., 1996. Vac Res 5:91-102; Zimmerman et al., 1996. Vac Res 5:103-13; Zimmerman et al., 1985. DNA. Med Virol 15:215-22. Charoenvit et al., 2004. Antimicrob Agents Chemother 48:2455; Charoenvit et al., 2004. Vaccine 10:2368; and Zimmerman et al., 2001. Vaccine 19(32):4750-4759). The I/TCBLs are generally derived from immune system molecules known or suspected to bind to T cells, examples of which include portions of MHC Classes I and II or accessory molecules such as β2-microglobulin, portions of LFA-3, portions of the Fc region of the heavy chain of immunoglobulins, and Ia+ molecules. In specific embodiments, β2 microglobulin (J) is used as the I/TCBL and linked to an autoantigen of interest. Fusion constructs of this type have been used successfully to arrest disease progression in a collagen-induced arthritis model, as described for example by Zimmerman et al. (2010, Int Immunopharmacol 10:412-421) with increased IL-12p70 and IL-10 as well as reduced IL-17 and TNFα in the serum of treated animals.
Exemplary methods for making L.E.A.P.S. constructs are disclosed for example in U.S. Pat. No. 5,652,342 and U.S. Pat. Appl. No. 2011/0098444, which are hereby incorporated by reference herein in their entirety.
3.3 Cell Based Therapy or ProphylaxisAggrecan-specific antigen-presenting cells as described for example in Section 3.2.7, or aggrecan-specific T or B regulatory lymphocytes or tolerogenic B lymphocytes as described for example in Section 3.2.9 can be administered to a patient, either by themselves or in combination, for suppressing an immune response to an aggrecan antigen, including citrullinated forms thereof. These cell based compositions are useful for treating or preventing joint damage in affected or predisposed subjects including subjects with early RA and subjects at risk of developing RA, suitably before the disease progresses to the chronic and debilitating form of RA. The cells of the invention can be introduced into a patient by any means (e.g., injection), which produces an antigen-specific tolerogenic response to aggrecan antigen. The cells may be derived from the patient (i.e., autologous cells) or from an individual or individuals who are MHC matched or mismatched (i.e., allogeneic) with the patient. Typically, autologous cells are injected back into the patient from whom the source cells were obtained. The injection site may be subcutaneous, intraperitoneal, intramuscular, intradermal, intravenous or intralymphoid. The cells may be administered to a patient already suffering from joint damage (including subjects with early RA and subjects at risk of developing RA) or who is predisposed to joint damage in sufficient number to result in a clinical improvement in the subject or to prevent joint damage or the symptoms of joint damage in a subject at risk of developing joint damage (including subjects at risk of developing RA). The number of cells injected into the patient in need of the treatment or prophylaxis may vary depending on inter alia, the aggrecan antigen or antigens and size of the individual. This number may range for example between about 103 and 1011, and usually between about 105 and 107 cells (e.g., in the form blood, PMBC or purified dendritic cells, T lymphocytes, hematopoietic or lymphoid cells, B lymphocytes etc.). Single or multiple (2, 3, 4 or 5) administrations of the cells can be carried out with cell numbers and pattern being selected by the treating physician. The cells should be administered in a pharmaceutically acceptable carrier, which is non-toxic to the cells and the individual. Such carrier may be the growth medium in which the cells were grown, or any suitable buffering medium such as phosphate buffered saline. The cells may be administered alone or as an adjunct therapy in conjunction with other therapeutics known in the art for the treatment or prevention of unwanted immune responses for example but not limited to glucocorticoids, methotrexate, D-penicillamine, hydroxychloroquine, gold salts, sulfasalazine, TNFα or IL-1 inhibitors, and/or other forms of specific immunotherapy.
3.4 Pharmaceutical CompositionsIn accordance with the present invention, aggrecan antigens described for example in Section 3.2.1, aggrecan APL as described for example in Section 3.2.2, NF-κB inhibitors as described for example in Section 3.2.3, mTOR inhibitors as described for example in Section 3.2.4, Syk inhibitors as described for example in Section 3.2.5, immunomodulating particles described for example in Section 3.2.6, antigen-presenting cells as described for example in Section 3.2.7, regulatory T or B lymphocytes or tolerogenic B lymphocytes as described for example in Section 3.2.9, MHC-peptide complexes as described for example in Section 3.2.10, and chimeric constructs described for example in Section 3.2.11 (also collectively referred to herein as “immune modulators”) are useful in compositions and methods for suppressing an immune response to an aggrecan antigen, including citrullinated forms thereof, and are especially useful for treating or preventing joint damage in affected or predisposed subjects including subjects with early RA and subjects at risk of developing RA.
The immune modulator-containing compositions of the present invention are typically in the form of pharmaceutical compositions, which may comprise a pharmaceutically acceptable carrier or diluent. Depending on the specific conditions being treated, the immune modulator may be formulated and administered systemically, topically or locally. Techniques for formulation and administration may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition. Suitable routes may, for example, include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections. For injection, the immune modulators of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. Intra-muscular and subcutaneous injection is appropriate, for example, for administration of immunogenic compositions, vaccines and DNA vaccines.
The immune modulators can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration. Such carriers enable the compounds of the invention to be formulated in dosage forms such as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. These carriers may be selected from sugars, starches, cellulose and its derivatives, malt, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline, and pyrogen-free water.
Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
Pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association one or more immune modulators as described above with the carrier, which constitutes one or more necessary ingredients. In general, the pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
Pharmaceutical which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added.
Dosage forms of the drugs of the invention may also include injecting or implanting controlled releasing devices designed specifically for this purpose or other forms of implants modified to act additionally in this fashion. Controlled release of an immune modulator of the invention may be effected by coating the same, for example, with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids and certain cellulose derivatives such as hydroxypropylmethyl cellulose. In addition, controlled release may be achieved by using other polymer matrices, liposomes or microspheres.
The immune modulators of the present invention may also be administered to the respiratory tract as a nasal or pulmonary inhalation aerosol or solution for a nebulizer, or as a microfine powder for insufflation, alone or in combination with an inert carrier such as lactose, or with other pharmaceutically acceptable excipients. In some particulate embodiments of the present invention, the particles of a formulation may advantageously have diameters of less than 50 μm, suitably less than 10 μm.
In some particulate embodiments, the immune modulators are administered for active uptake by APC, for example by phagocytosis, as described for example in U.S. Pat. No. 5,783,567 (Pangaea). The phagocytosis by these cells may be improved by maintaining a particle size typically below about 20 μm, and preferably below about 11 μm.
In specific particulate embodiments, immune modulators in particulate form are delivered directly into the bloodstream (i.e., by intravenous or intra-arterial injection or infusion) if uptake by the phagocytic cells of the reticuloendothelial system (RES), including liver and spleen, is desired. Alternatively, one can target, via subcutaneous injection, take-up by phagocytic APC of the draining lymph nodes. Particles can also be introduced intradermally (i.e., to the APCs of the skin, such as dendritic cells and Langerhans cells) for example using ballistic or microneedle delivery. Illustrative particle-mediated delivery techniques include explosive, electric or gaseous discharge delivery to propel carrier particles toward target cells as described, for example, in U.S. Pat. Nos. 4,945,050, 5,120,657, 5,149,655 and 5,630,796. Non-limiting examples of microneedle delivery are disclosed in International Publication Nos. WO 2005/069736 and WO 2005/072630 and U.S. Pat. Nos. 6,503,231 and 5,457,041.
In other specific particulate embodiments, the route of particle delivery is via the gastrointestinal tract, e.g., orally. Alternatively, the particles can be introduced into organs such as the lung (e.g., by inhalation of powdered microparticles or of a nebulized or aerosolized solution containing the microparticles), where the particles are picked up by the alveolar macrophages, or may be administered intranasally or buccally. Once a phagocytic APC phagocytoses the particle, the NF-κB inhibitor and optionally the aggrecan antigen are released into the interior of the cell.
Suitable routes for particle delivery include for example oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections. For injection, particles may be formulated in aqueous solutions, suitably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
Pharmaceutical formulations for parenteral administration of particles include aqueous solutions of particles in water-soluble form. Additionally, suspensions of the particles may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
Exemplary methods for particulate delivery of inhibitor (e.g., NF-κB inhibitor) and antigen are described for example in U.S. Pat. Appl. Pub 20100151000.
The immune modulators of the invention (e.g., inhibitors(s), antigen, APCs, lymphocytes, fusion proteins, particles etc.) may be provided as salts with pharmaceutically compatible counterions. Pharmaceutically compatible salts may be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents that are the corresponding free base forms.
Pharmaceutical compositions suitable for use in the present invention include compositions wherein the immune modulators are contained in an effective amount to achieve their intended purpose. The dose of an immune modulator administered to a patient should be sufficient to achieve a clinical improvement in the subject or to prevent joint damage or the symptoms of joint damage in a subject at risk of developing joint damage (including subjects at risk of developing RA). The quantity or dose frequency of the immune modulator(s) to be administered may depend on the subject to be treated inclusive of the mode of administration, age, sex, weight and general health condition thereof. In this regard, precise amounts of the immune modulator(s) for administration will depend on the judgment of the practitioner. In determining the effective amount of the immune modulator(s) to be administered in the treatment or prophylaxis of joint damage, the practitioner may evaluate inflammation, pro-inflammatory cytokine levels, lymphocyte proliferation, cytolytic T lymphocyte activity and regulatory T lymphocyte function that is specific to aggrecan. In any event, those of skill in the art may readily determine suitable dosages of immune modulator(s).
For any immune modulator used in the methods of the present invention, the therapeutically effective dose can be estimated initially from cell culture assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC50 as determined in cell culture (e.g., the concentration of a test agent, which achieves a half-maximal reduction in NF-κB activity or mTOR activity or Syk activity, a maximal increase in aggrecan-specific antigen-presenting cells etc.). Such information can be used to more accurately determine useful doses in humans.
Toxicity and therapeutic efficacy of such immune modulators can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50 Immune modulators that exhibit large therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage of such immune modulators lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See for example Fingl et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch. 1 p 1).
Dosage amount and interval may be adjusted individually to provide plasma levels of the immune modulator, which are sufficient to enhance suppression of an immune response or tolerogenesis to an aggrecan antigen. Usual patient dosages of non-cellular immune modulators of the present invention for systemic administration range from 0.0001-2000 mg/day, commonly from 0.0001-250 mg/day, and typically from 0.001-150 mg/day.
The compositions of the invention may be administered over a period of hours, days, weeks, or months, depending on several factors, including the severity of the condition (e.g., joint damage including in RA) being treated, whether a recurrence of the condition is considered likely, etc. The administration may be constant, e.g., constant infusion over a period of hours, days, weeks, months, etc. Alternatively, the administration may be intermittent, e.g., immune modulators may be administered once a day over a period of days, once an hour over a period of hours, or any other such schedule as deemed suitable. In specific embodiments, the immune modulators of the present invention are administered on a regular regimen such as weekly, biweekly, monthly, quarterly, semi-annually or annually by one of the following routes: intradermally, intramuscularly or subcutaneously as well as cutaneous transdermal or nasal delivery in amounts of from 1-100, usually 10-50, micrograms per kilogram of body weight.
3.5 Methods for Assessing Antigen-Specific TolerogenesisThe efficiency of inducing effector lymphocytes, especially effector T lymphocytes, to exhibit tolerance to an aggrecan antigen can be determined by assaying immune responses to that antigen including, but not limited to, assaying effector T lymphocyte cytolytic activity in vitro using for example aggrecan-specific antigen-presenting cells as targets of antigen-specific cytolytic T lymphocytes (CTL); assaying aggrecan-specific T lymphocyte proliferation, apoptosis or cytokine production (see, e.g., Vollenweider and Groscurth, 1992. J. Immunol. Meth. 149:133-135), antigen-specific suppression of effector T cells, measuring B cell antibody response to the antigen using, for example, ELISPOT assays, and ELISA assays; interrogating cytokine profiles; or measuring delayed-type hypersensitivity (DTH) responses by test of skin reactivity to a specified antigen (see, e.g., Chang et al. 1993, Cancer Res. 53, 1043-1050).
In some embodiments, the antigen-specific tolerance/anergy induced by the aggrecan-specific regulatory T lymphocytes reflects the inability of the antigen-specific immune effector cells (e.g., antigen-specific effector T and/or B lymphocytes) to respond to subsequent restimulation with the aggrecan antigen. These antigen-specific regulatory lymphocytes may be characterized by production of cytokines, such as IL-10 or IFN-γ in an antigen-specific manner. IL-10 and IFN-γ are cytokines with potent immunosuppressive properties produced by CD25− CD127dim CD4+ induced regulatory T cells in humans. Thus, in some embodiments, the presence of anergic T lymphocytes may be determined by assaying cytokine production using standard assays known in the art, such as intracellular staining (Haringer et al., 2009. J Exp Med 206(5):1009-1017). Alternatively there may be changes in the proportion of CD4+ regulatory T cells expressing CD25 and FoxP3, which are CD127dim (Fazekas de St Groth B et al., 2011. Methods Mol Biol 707:263-79).
Alternatively, or in addition, antigen-specific tolerance/anergy may be determined indirectly by giving the treated subject at least about one month after administration of the immune modulator/tolerogenic composition, a radiographic test that measures alleviation or healing of the joint damage, or slowing down of the progression of joint damage, including its signs and symptoms and/or structural damage, as compared to baseline prior to the administration, wherein the amount of immune modulator/tolerogenic composition administered is effective in achieving alleviation or healing of the joint damage, or slowing down of the progression of joint damage, including its signs and symptoms and/or structural damage, indicating that the subject has been successfully treated for the joint damage. In specific embodiments, the radiographic testing after administering the immune modulator/tolerogenic composition occurs at least about two months, at least about 10 weeks, at least about three months, at least about four months, at least about five months, at least about 24 weeks, at least about six months, or at least about 52 weeks after administering the immune modulator/tolerogenic composition. In illustrative examples of this type, the test measures a total modified Sharp score.
In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non-limiting examples.
EXAMPLES Example 1 T Cells Proliferate Poorly but Produce Cytokines in Response to Citrullinated Autoantigenic PeptidesCitrullinated or unmodified peptide antigens were synthesized from the fibrinogen, vimentin, collagen and aggrecan protein sequences which had been identified, either based on predicted binding capacity to RA-associated DR molecules in a molecular model positioning citrulline at P4, or through previous studies in HLA-DR4-IE-transgenic mice (Table 5) (Hida et al., 2004. J. Autoimmun. 23:141-150; Hill J et al., 2008. J. Exp. Med. 205:967-979; von Delwig et al., 2010. Arthritis Rheum. 62:143-149).
In total, 21 SE+ RA patients and 6 SE+ healthy controls were studied. All except 4 RA patients were also ACPA+ (Table 6). Forty three percent of the RA patients were non-smokers, 38% past and 19% current smokers. One healthy control is a current smoker, and 2 have a family history of RA. The proliferative response of peripheral blood mononuclear cells (PBMC) was analyzed from SE+ RA patients and SE+ healthy controls to varying concentrations of citrullinated or unmodified peptide antigen. Proliferative responses to peptide antigens and tetanus toxoid were expressed as SI. SI>2 were considered significant. Mean proliferative SI to citrullinated and unmodified peptides were generally between 1 and 2 in RA patients and healthy controls, and significantly lower in each case than responses to tetanus toxoid (
In initial experiments, the present inventors determined that background cytokine secretion and proliferative responses were generally lower if peptide responses were assayed in the presence of human rather than fetal calf serum. Moreover, there was no difference in cytokine production when comparing assays carried out in the presence of 10% healthy or autologous or allogeneic RA donor serum, provided Hetero Block was added where rheumatoid factor (RF) titres were >100 U/mL in order to prevent RF from binding capture and detection antibodies in ELISA reactions (Todd et al., 2011. Arthritis & Rheumatism 63:894-903). In the absence of peptide stimulation, RA patient PBMC secreted significantly higher concentrations of IFN-γ and IL-6 than other cytokines in the absence of peptides (
To better understand the citrullinated peptide response pattern of individual RA patients and SE+ healthy controls, IL-6 dose response curves were plotted for each peptide for each individual in the study. PBMC from 4/6 healthy controls dose-dependently secreted IL-6 to citrullinated aggrecan and 3/6 secreted IL-6 in response to citrillated fibrinogen. Taking the same threshold for a positive response as described above, it was found that of 17 RA patients' PBMC responses, 6 secreted IL-6 in response to no epitopes, 8 to citrullinated aggrecan only, 0 to citrullinated fibrinogen, 0 to citrullinated collagen type II only and 3 to multiple citrullinated epitopes. Thus, IL-6 responses were highest and observed most frequently towards citrullinated aggrecan in both RA patients and healthy controls, suggesting this was the most immunogenic epitope tested. An IL-6 response to multiple citrullinated epitopes occurred more frequently among patients diagnosed with RA at least 5 years previously (
IL-6 is an important cytokine in RA, which could be produced upon stimulation of PBMC either by T cells or antigen presenting cells. To determine the origin of the cytokines secreted into supernatants of PBMC stimulated by citrullinated peptides, RA PBMC were incubated with citrullinated or native peptides for 5 days, with addition of Brefeldin-A for the last 18 hours, prior to intracellular cytokine staining along with analysis of cell surface markers. CD3+CD4+ T cells produced more intra-cellular IFN-γ and IL-6 when incubated with citrullinated aggrecan or fibrinogen relative to incubation in medium alone (
The present inventors have shown herein that pro-inflammatory and regulatory cytokines, including IL-6, IFN-γ IL-10 and TNF were produced by CD4+ T cells in SE+ RA patients in response to citrullinated self-epitopes, of which citrullinated aggrecan was most immunogenic. These cytokine responses were observed in spite of weak peptide-specific T cell proliferative responses. SE+ healthy controls also produced cytokines in response to citrullinated aggrecan and citrullinated fibrinogen. Such T cell responses are not necessarily causally related to RA—rather they demonstrate the autoreactivity present in SE+ individuals towards citrullinated self-peptides, even in the absence of ACPA. Cytokine responses to citrullinated self-epitopes were, however, more diverse in RA than healthy control individuals. Indeed only RA patients secreted the regulatory cytokines IL-10 and IFN-γ in response to these epitopes (Haringer et al., 2009. J Exp Med 206(5):1009-1017). IFN-γ production by PB and synovial T cells is well-described (Steiner et al., 1999. Rheumatology 38(3):202-213), and IFN-regulated genes have been shown to be predictive of RA development in ACPA+ patients with arthralgia (van Baarsen et al., 2010. Arthritis & Rheumatism 62(3):694-704). By intra-cellular staining the present inventors demonstrated IL-6 and IFN-γ production by memory CD4+ T cells but not by CD4− antigen-presenting cells in these cultures, at least when examined after 5 days of peptide stimulation. These data demonstrate that CD4+ T cells were capable of cytokine production but do not exclude the possibility that CD4− antigen presenting cells such as moncytes, B cells and dendritic cells could also produce these cytokines during the culture, which could be secreted into the supernatant. IL-6 responses increased dose-dependently in response to citrullinated peptides. Intracellular cytokine staining confirmed a broad cytokine response profile, consistent with the phenotype of memory CD4+ T cells.
Various mechanisms could contribute to the low proliferative response exhibited by autoreactive T cells to self-peptides and tetanus toxoid antigen in RA. For instance, a number of studies implicate deficient signaling of RA T cells through the T cell receptor-CD3 complex (Emery et al., 1984. Clin. Exp. Immunol. 57:123-129; Seitz et al., 1988. Rheumatol. Int. 8:189-196; Allen et al., 1995. Eur. J. Immunol. 25:1547-1554; Thomas et al., 1992. Arthritis Rheum. 35:1455-1465; Berg et al., 2000. Arthritis Res. 2:75-84; Maurice et al., 1997. J. Immunol. 159:2973-2978; Berg et al., 2000. Clin. Exp. Immunol. 120:174-182). IL-2 might be rapidly consumed through binding to the high avidity CD25 receptor expressed by regulatory T cells and by effector memory T cells upon activation. This could limit availability of IL-2 for T cell proliferation in tissue culture (Wolf et al., 2001. Eur. J. Immunol. 31:1637-1645; Ishimaru et al., 2006. Nat. Immunol. 7:763-772). Furthermore, autoreactive T cells are subject to the influence of regulatory cells and cytokines, as demonstrated here (Berg et al., 2001. Ann. Rheum. Dis. 60:133-139; van Amelsfort et al., 2004. Arthritis Rheum. 50:2775-2785). On the other hand, effector memory cells produce high levels of pro-inflammatory and regulatory cytokines, and cytokine production more effectively interrogates highly differentiated autoreactive effector memory T cells in both humans and mice (Garcia de Tena et al., 2006. J. Clin. Immunol. 26:233-242; Nanki et al., 2000. Arthritis Res. 2:415-423; Morita et al., 1998. Arthritis Rheum. 41:1669-1676).
The pro-inflammatory cytokine IL-6 stimulates B cell antibody production and plays a critical role in RA pathogenesis (Assier et al., 2010. Joint Bone Spine 77:532-536). In RA patients, intracellular IL-6 and IFN-γ were produced by CD28+ and CD28− and CD45RO+ and CD45RO− PB CD4+ T cells. Consistent with the poor proliferative but good cytokine response of RA T cells in response to citrullinated peptides in the current studies, CD4+CD45RBdimCD27− memory T cells from healthy donors were previously shown to proliferate poorly but to produce large amounts of IL-4 and IL-10 in response to mitogen, and to provide effective B cell help for immunoglobulin production (Tortorella et al., 1995. J. Immunol. 155:149-162). In contrast, typical CXCR5+ follicular helper T cells, which have been shown to promote antibody production in lymphoid tissue in vivo (Chevalier et al., 2011. J Immunol 186:5556-5568), did not express cytokines in response to citrullinated peptides. Of interest, where CD28− T cells were present in PB of RA patients, they also produced cytokine in response to citrullinated peptides. CD28− T cells represent an important effector memory amplification response in the synovial environment, and have been shown to be more resistant to suppression by regulatory T cells (Weng et al., 2009. Trends Immunol. 30:306-312; Thewissen et al., 2007. J. Immunol 179:6514-6523). Together the data presented herein support the hypothesis that effector memory T cells reactive with a variety of citrullinated self-peptides and with the potential for B cell help circulate in peripheral blood and are present in synovial fluid of SE+ individuals.
Previous studies in RA patients analyzed the PB CD4+ T cell response to a single citrullinated peptide specificity. Proliferative and IL-17 CD4+ T cell responses to citrullinated aggrecan 84-103 were demonstrated in RA patients but not in healthy controls, demonstrating the immunogenicity of this citrullinated aggrecan epitope (von Delwig et al., 2010. Arthritis Rheum 62(1):143-149). The unmodified aggrecan epitope is immunodominant in BALB/c mice immunized with aggrecan/proteoglycan (Buzás et al., 2005. Cell Immunol 235(2):98-108). This citrullinated aggrecan peptide was also the most immunogenic epitope in the current studies—with the highest frequency of responders, and the highest magnitude of IL-6 responses. It should be noted, however, that the Buzás study did not compare early RA and longstanding RA patients, nor did it investigate the host immune response to any other antigen beyond aggrecan.
In long-standing RA patients, the present inventors also found robust cytokine production was also found in response to citrullinated fibrinogen-α 79-91, which was shown to be the immunodominant epitope in HLA-DR4-IE transgenic mice immunized with citrullinated human fibrinogen (Hill et al., 2008. J. Exp. Med. 205:967-979). Stimulation with the citrullinated vimentin 66-78 epitope produced much weaker cytokine responses in the current studies, as was observed by Snir et al. (2011, supra), who found that HLA-DRB1*0401+ RA patient T cells produced a number of cytokines when incubated with citrullinated vimentin 59-78, but not 66-78. In that study, IFN-γ and TNF were expressed intracellularly by a small proportion of CD154+ CD4+ RA patient T cells in response to citrullinated but not native vimentin 59-78. The proportion of T cells staining for intracellular cytokine was much lower than in the current studies, in which the present inventors carried out staining 5 days after antigen stimulation and without a second restimulation. It is possible that anergy may have been induced by restimulation with peptide after an initial 5 day culture with peptide, or given their effector memory phenotype (Di Mitri et al. 2011. “Reversible Senescence in Human CD4+CD45RA+CD27—Memory T Cells.” J. Immunol), that few T cells were capable of re-expressing CD154 after prolonged in vitro culture. Similar to the current study, Snir et al. (2011, supra) did not observe strong proliferative or IL-17A responses, unlike von Delwig et al. (2010, supra). Differences in cytokine secretion may relate to the inclusion of healthy human serum or of Hetero Block plus autologous serum in the current studies (Todd et al., 2011. Arthritis & Rheumatism 63:894-903). Finally, due to the hydrophobic amino acids at 84-88, the citrullinated aggrecan peptide was relatively insoluble in aqueous medium, which may also have affected antigen purity, concentration and cellular uptake in different laboratories.
The present inventors also observed T cell cytokine responses to citrullinated self-epitopes in SE+ RA patients, whether ACPA+ or not, as well as in ACPA− healthy controls. HLA-DRB1*0401+ healthy control T cells were also observed to produce cytokine in response to citrullinated vimentin 59-78 (Snir et al., 2011, supra). After immunization of DR4-IE transgenic mice with citrullinated human fibrinogen, responses to unmodified native epitopes were stimulated in parallel with responses to citrullinated epitopes. From the previous and current analysis of HLA-DR binding and responses to unmodified epitopes, responses to citrullinated epitopes appear to be most specific to SE+ RA PB. In contrast, the magnitude and diversity of the responses to citrullinated epitopes in healthy controls was unexpected. There was no T cell autoreactivity towards citrullinated fibrinogen in DR4-IE transgenic mice primed with native human fibrinogen (Hill et al., 2008, supra). Spontaneous autoreactivity towards citrullinated fibrinogen in naïve or stressed DR4-IE-transgenic mice has not been tested. However, it has been shown that CD4+ T cells in both SE+ RA patients and HLA-DR4+ healthy controls have reduced T cell receptor excision circles, overall telomere shortening, and reduced replicative capacity, which together imply a HLA-DR SE-associated reduction in T cell input to the peripheral repertoire, an increased proliferative drive for naïve T cells towards peripheral self-antigens and a limited diversity of the TCR repertoire (Fujii et al., 2009. Proc Natl Acad Sci USA 106:4360-4365; Koetz et al., 2000. Proc Natl Acad Sci USA 97:9203-9208; Schönland et al., 2003. Proc Natl Acad Sci USA 100:13471-13476). The current and previous studies in humans suggest together that PB T cells in HLA-DR SE+ individuals may be predisposed to autoreactivity towards self-antigens including those modified by citrullination, potentially exposed during stress or pro-inflammatory settings, including joint trauma or smoking (Klareskog et al., 2006. Arthritis Rheum. 54:38-46; de Jong et al., 2010. Ann. Rheum. Dis. 69:255-262). Furthermore, unlike T cell autoreactivity, the development of B cell autoreactivity towards citrullinated epitopes appears to be a significant checkpoint in the progression to RA. It has been proposed that infection e.g. with Porphyromonas gingivalis in patients with periodontitis, may be a critical trigger for progress through this checkpoint, potentially due to cross reactivity with bacterial antigens, adjuvant effects of pathogen-associated molecular patterns, targeting of alternative antigen presenting cells, or a combination of these (Wegner et A L., 2010. Immunol. Rev. 233:34-54). Carbamylation of lysine residues to homocitrulline may be another factor enhancing the immunogenicity of RA autoantigens, including citrullinated antigens (Mydel et al., 2010. J Immunol. 184:6882-6890).
The data presented herein indicate that RA patients with long-standing disease are more likely to respond to multiple citrullinated epitopes, whereas patients with recent-onset RA (including those previously untreated) are more likely to respond either to no antigen or only to citrullinated aggrecan. These data also indicate that epitope spreading occurs as disease progresses. The assay developed herein, in which T cell cytokine production is interrogated in the presence of varying concentrations of a panel of citrullinated epitopes is proposed to be a useful biomarker to identify the most immunogenic peptide epitopes, and to correlate specific T cell responses with corresponding ACPA “fine specificities” (Willemze et al., 2011. Arthritis Rheum. 63:1823-1832). Advantageously, these assays can be carried out in conjunction with analysis of tetramer-specific T cells. Contemplated applications include comparison of serial samples of PBMC from pre-RA through to diagnosis (Deane et al., 2010. Arthritis Rheum. 62:3161-3172), and analysis of PBMC from clinical trials of antigen-specific or non-specific immunotherapy, such as Abatacept (Yue et al., 2010. Arthritis Rheum. 62:2941-2952). Finally, analysis of specific peptide reactivity is proposed to be beneficial for stratifying patients and for identifying appropriate epitopes for personalized antigen-specific immunotherapy.
Materials and Methods PatientsTwenty one patients who fulfilled the 1987 American College of Rheumatology (ACR) criteria for RA (Aletaha et al., 2010, supra) and 6 non-smoking ACPA− SE+ healthy controls were included. All individuals provided peripheral blood (PB) samples, although in some cases the yield was insufficient for all assays. Patient demographic details are outlined in Table 6. HLA-DR genotyping was carried out at Queensland Health Pathology Services. The study was approved by the Human Research Ethics Committee of the Princess Alexandra Hospital and informed consent was obtained from each patient.
Citrulline or arginine-containing SE-binding epitopes corresponding to vimentin, collagen type II, fibrinogen and aggrecan were synthesized by Auspep (NSW, Australia). All peptides were filtered, reconstituted to 300 μg/mL in sterile water and stored at −70° C. Final dilutions for working stock were made with medium.
Purification of PB Mononuclear Cells and Antigen Presentation AssaysMononuclear cells (MC) were isolated from PB and SF using Ficoll-Paque density gradients (Amersham Pharmacia Biotech, Uppsala, Sweden) and washed with 0.9% saline then resuspended in RPMI and 10% human serum from healthy or autologous or allogeneic RA donors. Tetanus toxoid was used at a concentration of 4 Lfi/mL (Chiron Vaccinese). Two×105 PBMC or SFMC were incubated with 0, 3 and 30 μg/mL of each peptide in the presence of RPMI containing 10% human serum in a final volume of 200 μL in round-bottomed wells, for 5 days. T cell proliferation was assessed by addition of 1 μCi/well [3H]thymidine (ICN Biochemicals) for the final 18 hours. Cells were harvested onto glass fibre filter mats and [3H]-thymidine incorporation was determined by liquid scintillation spectroscopy (Packard Topcount, Packard Instrument Co.). IL-2, IL-4, IFNγ, IL-10, IL-6, IL-17 and TNF were measured in day 5 supernatants using BD Cytometric Bead Array (CBA) kits (BD Bioscience). Initial kinetic experiments demonstrated that peptide stimulation for 5 days optimally induced antigen-specific proliferative and cytokine responses, and that longer cultures did not yield greater responses. Where RA serum was used in cytokine production assays, 150 μg/mL HeteroBlock (Omega Biologicals) were added during CBA measurement (Todd et al., 2011, supra). Samples were read on the BD FACSArray™ bioanalyzer system. Stimulation indices (SI) for proliferative responses were calculated as fold increase in response to peptide over background. Net cytokine secretion was calculated for each response as [concentration with peptide stimulation] minus [concentration without peptide stimulation]. Positive response thresholds were 2 SD above the cytokine responses towards the corresponding native peptide, for both RA patients and healthy controls.
Flow Cytometry and Intracellular Cytokine StainingPBMC from SE+ RA patients or healthy controls were incubated with or without peptides at concentrations of 3 and 30 μg/mL for 5 days, with Brefeldin A (Sigma Aldrich) added for the final 18 hours. Cells were stained for CD3-FITC, CD4-APC/Cy7, CD28-FITC, CXCR5-PerCP/Cy5.5 and CD45RO-PerCP/Cy5.5 (Biolegend), followed by permeabilization and staining for intracellular IL-6-APC and IFN-γ-APC (Biolegend). Data were collected on the Gallios flow cytometer and analysed using Kaluza software (Beckman Coulter).
Statistical AnalysisOne-way non-parametric ANOVA with post-hoc correction compared multiple means. Unpaired Mann Whitney tests compared the tetanus toxoid proliferative responses between RA patients and healthy controls and specific cytokine responses to citrullinated vs native peptides. Significance is indicated as *P<0.05, **P<0.005, and **P<0.001. All error bars represent SEM.
Example 4 Inhibition of APC from an RA Patient with Inhibitors of NF-κB, mTOR or Syk Suppresses their Capacity to Induce T Cell Cytokine Production in Response to Citrullinated Aggrecan PeptidePeripheral blood mononuclear cells (PBMC) were extracted from an RA patient with disease duration 18 years, positive anti-CCP and HLA-DRB1*0401 shared epitope. Autologous CD4+ T cells were isolated using immunomagnetic beads. The non-T cell fraction consists of antigen-presenting cells (APC), including dendritic cells, monocytes and B cells; it was treated with Mitomycin C to prevent proliferation. The APC were pre-incubated for 1 h without or with 3 uM BAY7011-82, 20 μg/mL Curcumin (both inhibit NF-kB), 10 ng/mL Rapamycin (inhibits mTOR) or 4 0 μM Piceatannol (inhibits syk), then incubated with 100 ng/mL LPS for 1 h. The APC were washed 3 times then incubated for 5 days with CD4+ T cells in the presence of 0 μg/mL, 3 μg/mL or 30 μg/mL Aggrecan peptide 84-103 cit 93. Cytokines were analysed in culture supernatants by cytokine bead array. The data show net (peptide minus no peptide wells) IL-6 and TNF production in response to 3 or 30 μg/mL peptide.
The results presented in
PBMC from the same RA patient as described in Example 4 were incubated with the native or citrulinated form of the 351 amino acid human aggrecan G1 epitope (contains dominant and subdominant epitopes in mice), for 5 days at 1 μg/mL, then IL-6 was measured in supernatant. Net IL-6 (antigen minus no antigen wells) production shown in
PBMC from 2 HLA-DRB1*0401+ ACPA+ RA patients were stained with CLIP, influenza hemagglutinin (HA), aggrecan 89-103 cit 93, 95 or vimentin 59-71 cit 64, 69, 71 DRB1*0401-tetramers in the presence of dasatinib. Plots were gated on live cells, CD4+ T cells, enriched for tetramer+ cells using immunomagnetic beads. The % tetramer+ of enriched PBMC is shown in
PBMC from 6 RA patients and 3 healthy controls were stained as in Example 6 with inclusion of fluorescent counting beads, then the number of tetramer+ T cells was calculated per mL blood. The mean fluorescence intensity (MFI) of tetramer staining is an indicator of affinity of the T cell receptor for the antigen presented by the HLA-DR molecule. Cit-aggrecan-specific T cells circulate in RA patients and healthy controls of this genotype. The results presented in
The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety.
The citation of any reference herein should not be construed as an admission that such reference is available as “Prior Art” to the instant application.
Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. Those of skill in the art will therefore appreciate that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention. All such modifications and changes are intended to be included within the scope of the appended claims.
Claims
1. A method for treating or preventing joint damage in a subject with early RA or incipient RA, the method comprising eliciting an antigen-specific tolerogenic response to an aggrecan polypeptide in the subject, thereby treating or preventing the joint damage.
2. The method according to claim 1, wherein the aggrecan polypeptide is a citrullinated aggrecan polypeptide.
3. The A method according to claim 1, further comprising identifying that the subject has early RA or incipient RA prior to eliciting the antigen-specific tolerogenic response.
4. The method according to claim 1, wherein the subject is positive for the shared epitope (SE).
5. The A method according to claim 4, further comprising identifying that the subject is positive for SE prior to eliciting the antigen-specific tolerogenic response.
6. The method according to claim 1, wherein the subject has or is at risk of developing an immune response to the aggrecan polypeptide.
7. method according to claim 6, wherein the immune response comprises an effector T lymphocyte response.
8. The method according to claim 7, wherein the immune response comprises production of at least one cytokine selected from the group consisting of interleukin-6 (IL-6), interferon-γ (IFN-γ), tumor necrosis factor (TNF), and interleukin-10 (IL-10).
9. The A method according to claim 6, wherein the immune response includes a pro-inflammatory T lymphocyte response, which comprises, consists or consists essentially of production of at least one cytokine selected from the group consisting of IL-6, IFN-γ and TNF.
10. The method according to claim 9, wherein the pro-inflammatory T lymphocyte response comprises, consists or consists essentially of production of IL-6.
11. The method according to claim 6, wherein immune response is produced at least in part by CD4+ lymphocytes.
12. A method according to claim 1, wherein the antigen-specific tolerogenic response is effected by:
- increasing the number of tolerogenic antigen-presenting cells (agg-tolAPC) in the subject, which present a peptide corresponding to a portion of the aggrecan polypeptide, wherein the portion is associated with a pro-inflammatory or autoreactive T lymphocyte response to the aggrecan polypeptide;
- inducing anergy or apoptosis in pro-inflammatory or autoreactive T lymphocytes in the subject, which are reactive against the aggrecan polypeptide; and
- increasing the number of regulatory or suppressor T lymphocytes in the subject, which suppress or otherwise reduce a pro-inflammatory or autoreactive T lymphocyte response to the aggrecan polypeptide.
13. The method according to claim 12, comprising increasing the number of agg-tolAPC in the subject to thereby treat or prevent the joint damage.
14. The method according to claim 13, wherein the agg-tolAPC stimulate the production of regulatory or suppressor T lymphocytes that suppress or otherwise reduce the pro-inflammatory or autoreactive T lymphocyte response to the aggrecan polypeptide.
15. The method according to claim 13, wherein the agg-tolAPC are produced by contacting antigen-presenting cells in the subject with (1) an NF-κB inhibitor in an amount sufficient to inhibit NF-κB activity in the antigen-presenting cells, and/or (2) a mTOR inhibitor in an amount sufficient to inhibit mTOR activity in the antigen-presenting cells, and/or (3) a Syk inhibitor in an amount sufficient to inhibit Syk activity in the antigen-presenting cells, together with an antigenic molecule selected from an antigen that corresponds in whole, or in part, to an aggrecan polypeptide or a nucleic acid molecule from which the antigen is expressible, in an amount sufficient for the antigen-presenting cells to present the antigen or a processed form thereof on their surface.
16. The method according to claim 15, wherein the antigen comprises an amino acid sequence corresponding to a full-length aggrecan polypeptide.
17. The method according to claim 15, wherein the antigen comprises an amino acid sequence corresponding to a mature aggrecan polypeptide.
18. The method according to claim 15, wherein the antigen comprises an amino acid sequence corresponding to a domain of an aggrecan polypeptide selected from the G1 domain, the G2 domain, or the G3 domain.
19. The method according to claim 15, wherein the antigen comprises an amino acid sequence corresponding to a T cell epitope of an aggrecan polypeptide.
20. The method according to claim 19, wherein the amino acid sequence is selected from any one of SEQ ID NO: 5-35, including citrullinated forms thereof.
21. The method according to claim 19, wherein the amino acid sequence is selected from any one of SEQ ID NO: 32-35.
22. The method according to claim 15, wherein the antigen is HLA DR restricted and the subject is positive for an HLA DR allele.
23. The method according to claim 15, wherein the antigen is in the form of one or more peptides corresponding in whole or in part to an aggrecan polypeptide, including citrullinated forms thereof.
24. The method according to claim 15, wherein the antigen is in the form of a plurality of contiguous overlapping peptides whose sequences span at least a portion of an aggrecan polypeptide, including citrullinated forms thereof.
25. The method according to claim 15, wherein the overlapping peptides comprise, consist or consist essentially of an amino acid sequence selected from SEQ ID NO: 49-531, including citrullinated forms thereof.
26. The method according to claim 15, wherein the inhibitor is an NF-κB inhibitor (e.g., an NF-κB inhibitor selected from any one of the inhibitors listed in Tables 2, 3A, 3B or 4).
27. The method according to claim 26, wherein the NF-κB inhibitor is curcumin or a curcumin derivative.
28. The method according to claim 15, wherein the inhibitor and the antigenic molecule are co-administered in soluble form.
29. The method according to claim 15, wherein the inhibitor and the antigenic molecule are co-administered in particulate form.
30. The method according to claim 29, wherein the inhibitor and the antigenic molecule are co-administered in the same particle.
31. The method according to claim 29, wherein the particle is a polymeric particle.
32. The method according to claim 29, wherein the particle is a liposome.
33. The method according to claim 12, wherein the agg-tolAPC are produced by expressing in B lymphocytes a nucleic acid molecule that encodes an antigen that corresponds in whole, or in part, to an aggrecan polypeptide, wherein the expression of the nucleic acid molecule leads to presentation of the antigen or processed form thereof on the surface of the B lymphocytes.
34. The method according to claim 33, wherein the nucleic acid molecule further encodes an immunoglobulin or an immunoglobulin fragment fused directly or indirectly to the antigen.
35. The method according to claim 12, comprising administering to the subject regulatory or suppressor T lymphocytes, which suppress or otherwise reduce the pro-inflammatory or autoreactive T lymphocyte response to the aggrecan polypeptide.
36. The method according to claim 1, comprising administering to the subject a MHC-peptide complex consisting essentially of an antigen that corresponds in whole, or in part, to the aggrecan polypeptide and an isolated MHC component having an antigen-binding site, wherein the antigen is associated with the antigen-binding site.
37. The method according to claim 1, comprising administering to the subject a chimeric construct comprising an immunomodulatory peptide and an immune- or T cell-binding ligand (I/TCBL), wherein the immunomodulatory peptide comprises, consists or consists essentially of an amino acid sequence corresponding to a portion of an aggrecan polypeptide, and which binds to an antigen receptor on pro-inflammatory or autoreactive T lymphocytes, and wherein the I/TCBL binds to a class or subclass of T cell selected from the group consisting of helper T cells, suppressor T cells and cytotoxic T cells and modulates T cell activity.
38. The method according to claim 37, wherein the I/TCBL comprises at least a portion of a molecule selected from a MHC class I molecule, a MHC class II molecule, an accessory molecule such as β2-microglobulin, lymphocyte function associated molecule-3 (LFA-3), the Fc region of the heavy chain of an immunoglobulin molecule, Ia+ molecules, an antibody to CD2, an antibody to CD3, an antibody to CD4, an antibody to CD8, an antibody to lectin, a lymphokine.
39. A method of treating or preventing joint damage in a subject with early RA or incipient RA, comprising administering to said subject an agent or combination of agents that elicits an antigen-specific tolerogenic response to an aggrecan polypeptide.
40. The method according to claim 39, wherein the agent or combination of agents is selected from the group consisting of: (1) an inhibitor of the NF-κB pathway and an antigenic molecule selected from an antigen that corresponds in whole, or in part, to an aggrecan polypeptide or a nucleic acid molecule from which the antigen is expressible; (2) an mTOR inhibitor and an antigenic molecule selected from an antigen that corresponds in whole, or in part, to an aggrecan polypeptide or a nucleic acid molecule from which the antigen is expressible; (3) an inhibitor of the Syk pathway and an antigenic molecule selected from an antigen that corresponds in whole, or in part, to an aggrecan polypeptide or a nucleic acid molecule from which the antigen is expressible; (4) a nucleic acid molecule that encodes an antigen that corresponds in whole, or in part, to an aggrecan polypeptide for introduction into B lymphocytes; (5) a MHC-peptide complex consisting essentially of an antigen that corresponds in whole, or in part, to an aggrecan polypeptide and an isolated MHC component having an antigen-binding site, wherein the antigen is associated with the antigen-binding site; (6) a chimeric construct comprising an immunomodulatory peptide and an immune- or T cell-binding ligand (I/TCBL), wherein the immunomodulatory peptide comprises, consists or consists essentially of an amino acid sequence corresponding to a portion of an aggrecan polypeptide; (7) aggrecan-specific tolerogenic antigen-presenting cells; and (8) an APL that comprises an amino acid sequence corresponding to a portion of an aggrecan polypeptide.
41. (canceled)
Type: Application
Filed: Mar 25, 2013
Publication Date: Aug 6, 2015
Inventor: Ranjeny Thomas (Hawthorne)
Application Number: 14/387,529
